Methods and means for the production of IG-like molecules

ABSTRACT

The invention provides means and methods for producing one or more Ig-like molecules in a single host cell. Novel CH3 mutations enabling the production of monospecific and/or bispecific Ig-like molecules of interest are also provided.

This application is a Continuation of U.S. patent application Ser. No.13/866,747, filed Apr. 19, 2013, which claims priority to U.S.Provisional Patent Application No. 61/635,935, filed Apr. 20, 2012. Theentire contents of both of these patent applications, along with alldocuments cited therein, are hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 31, 2014, isnamed MRX5-007CN_Sequence_Listing.txt and is 8,489 bytes in size.

FIELD

The invention relates to the fields of molecular biology, medicine andbiological therapeutics. It particularly relates to the field oftherapeutic antibodies for the treatment of various diseases.

BACKGROUND

Many currently used biological therapeutics are isolated recombinant,human or humanized monoclonal antibodies that enhance the ability of thebody's immune system to neutralize or eliminate cells and/or moleculesinvolved in disease processes or to eradicate invading pathogens orinfectious agents. Monoclonal antibodies bind to a single specific area,or epitope, of an antigen and, for use in therapy, are often selectedfor a desirable functional property such as for example killing of tumorcells, blocking of receptor-ligand interactions or virus neutralization.Nowadays, there are about 30 FDA approved monoclonal antibodies, whichare typically produced at large quantities and their biophysical andbiochemical characteristics can be analyzed in great detail to ensurebatch-to-batch consistency, which facilitates regulatory acceptability.Despite these favorable characteristics, monoclonal antibodies haveseveral disadvantages, some of which relate to their monospecific natureand the complexity of diseases. Diseases processes are oftenmultifactorial in nature, and involve redundant or synergistic action ofdisease mediators or up-regulation of different receptors, includingcrosstalk between their signaling networks. Consequently, blockade ofmultiple, different factors and pathways involved in pathology mayresult in improved therapeutic efficacy. By nature of theirmonospecificity, monoclonal antibodies can only interfere with a singlestep within the complex disease processes which often does not have anoptimal effect. In addition to not fully addressing multiple aspects ofa disease process, it has become clear that targeting a single epitopeon a single cellular or soluble protein or pathogen often will notsuffice to efficiently treat, disease because the target epitope may nolonger be available for the monoclonal antibody to bind to and exert thedesired effect. As an example, tumor cells often escape from monoclonalantibody therapy by down-regulation, mutation or shielding of the targetepitope present on a growth factor receptor. By activating alternativereceptors and/or their ligands, tumor cells than may exploit a differentpath leading to continued growth and metastasis. Similarly, viruses andother pathogens frequently mutate and lose or shield the target epitope,thereby escaping monoclonal antibody treatment. Monoclonal antibodiesthat bind to a single epitope often do not recruit the full spectrum ofeffector mechanisms evoked by polyclonal antibodies, including, amongstother things, opsonization (enhancing phagocytosis of antigens), sterichindrance (antigens coated with antibodies are prevented from attachingto host cells or mucosal surfaces), toxin neutralization, agglutinationor precipitation (antibodies binding several soluble antigens causeaggregation and subsequent clearance), activation of complement andantibody-dependent cellular cytotoxicity (antibodies enable the killingof target cells by natural killer cells and neutrophils).

Polyclonal antibodies for therapeutic applications may be obtained frompooled human serum. Such serum-derived therapeutic polyclonal antibodiesmay for example be used to treat or prevent infections caused by virusessuch as the rabies virus, cytomegalovirus and respiratory syncytialvirus, to neutralize toxins such as tetanus toxin and botulinum toxin orto prevent Rhesus D allograft immunization. A more widespread use ofserum-derived polyclonal antibody preparations has been prevented by thefact that source plasma is only available for a limited range of targetssuch as infectious diseases and toxins. Moreover, the products arehighly dependent on donor blood availability, both in terms of quantityand suitability, resulting in considerable variation between batches. Inaddition, screening technologies fail to keep up with constantlyevolving viruses, thus, immunoglobulin products carry a potential riskof infectious disease transmission. Finally, the long process of bloodcollection, screening and immunoglobulin purification meansplasma-derived immunoglobulins are expensive to produce.

Mixtures of monoclonal antibodies may improve the efficacy of monoclonalantibodies while avoiding the limitations associated with serum-derivedpolyclonal antibodies. In the art, combinations of two human orhumanized monoclonal antibodies have been tested in preclinical modelsand in clinical trials (for example mixtures of 2 monoclonal antibodiesagainst the HER2 receptor, mixtures of 2 antibodies against the EGFRreceptor and, 2 monoclonal antibodies against the rabies virus).

In the art, it has been shown that combinations of 2 monoclonalantibodies may have additive or synergistic effects and recruit effectormechanisms that are not associated with either antibody alone. Forexample, mixtures of 2 monoclonal antibodies against the EGFR or HER2were shown to more potently kill tumor cells based on a combination ofactivities including enhanced receptor internalization, improvedblockade of signalling pathways downstream of the receptors as well asenhanced immune effector-mediated cytotoxicity. For combinationtherapies based on 2 monoclonal antibodies, the component antibodies maybe produced separately and combined at the protein level. A drawback ofthis approach is the staggering cost of developing the 2 antibodiesindividually in clinical trials and (partially) repeating that processwith the combination. This would lead to unacceptable cost of treatmentsbased on antibody combinations. Alternatively, the 2 recombinant celllines producing the component monoclonal antibodies may be mixed in afermenter and the resultant mixture of antibodies may be purified as asingle preparation (WO 2004/061104). A drawback of this approach is thepoor control over the composition and hence reproducibility of theresulting recombinant polyclonal antibody preparation, especially whenconsidering that such compositions may change over time as the cells arebeing cultured.

During the past decade, bispecific antibodies have emerged as analternative to the use of combinations of 2 antibodies. Whereas acombination of 2 antibodies represents a mixture of 2 differentimmunoglobulin molecules that bind to different epitopes on the same ordifferent targets, in a bispecific antibody this is achieved through asingle immunoglobulin molecule. By binding to 2 epitopes on the same ordifferent targets, bispecific antibodies may have similar effects ascompared to a combination of 2 antibodies binding to the same epitopes.Furthermore, since bispecific antibodies of the IgG format combine 2different monovalent binding regions in a single molecule and mixturesof 2 IgG antibodies combine 2 different bivalent binding molecules in asingle preparation, different effects of these formats have beenobserved as well. From a technological and regulatory perspective, thismakes development of a single bispecific antibody less complex becausemanufacturing, preclinical and clinical testing involve a single,molecule. Thus, therapies based on a single bispecific antibody arefacilitated by a less complicated and cost-effective drug developmentprocess while providing more efficacious antibody therapies.

Bispecific antibodies based on the IgG format, consisting of 2 heavy andtwo light chains have been produced by a variety of methods. Forinstance, bispecific antibodies may be produced by fusing twoantibody-secreting cell lines to create a new cell line or by expressingtwo antibodies in a single cell using recombinant DNA technology. Theseapproaches yield multiple antibody species as the respective heavychains from each antibody may form monospecific dimers (also calledhomodimers), which contain two identical paired heavy chains with thesame specificity, and bispecific dimers (also called heterodimers) whichcontain two different paired heavy chains with different specificity. Inaddition, light chains and heavy chains from each antibody may randomlypair to form inappropriate, nonfunctional combinations. This problem,known as heavy and light chain miss-pairings, can be solved by choosingantibodies that share a common light chain for expression as bispecific.But even when a common light chain is used, expression of two heavychains and one common light chain in a single cell will result in 3different antibody species, i.e. two monospecific ‘parental’ antibodiesand the bispecific antibody so that the bispecific antibody of interestneeds to be purified from the resulting antibody mixture. Althoughtechnologies have been employed to further increase the percentage ofbispecific antibodies in the mixtures of parental and bispecificantibodies and to decrease the percentage of miss-paired heavy and lightchains, there remains a need for bispecific formats that eliminate orminimize some of the disadvantages mentioned above. Taken together, theart provides a variety of technologies and methods for generatingmonoclonal antibodies, bispecific antibodies, mixtures of monoclonalantibodies, or mixtures of monospecific and bispecific antibodies thatcan subsequently be used for therapeutic application in patients.However, as discussed above, each of these existing technologies andmethods have their drawbacks and limitations. There is thus a need forimproved anchor alternative technologies for producing biologicaltherapeutics in the form of mixtures or bispecific approaches fortargeting multiple disease-modifying molecules

DESCRIPTION OF THE INVENTION

The invention provides methods and means for improved and/or alternativetechnologies for producing biological therapeutics in the form ofmixtures or bispecific approaches for targeting multipledisease-modifying molecules, as well as products and uses resulting fromthese methods and means.

Various approaches are described in the art in order to promote theformation of a certain bispecific antibody of interest, thereby reducingthe content of undesired antibodies in the resulting mixture.

For antibodies, it is well-known that the CH3-CH3 interaction is theprimary driver for Fc dimerization (Ellerson J R., et al., J. Immunol1976 (116) 510-517; Deisenhofer J. biochemistry 1981 (20) 2361-2370). Itis furthermore well-known that when two CH3 domains interact with eachother they meet in a protein-protein interface which comprises “contact”residues (also called contact amino acids, interface residues orinterface amino acids). Contact amino acids of a first CH3 domaininteract with one or more contact amino acids of a second CH3 domain.Contact amino acids are typically within 5.5 Å (preferably within 4.5 Å)of each other in the three-dimensional structure of an antibody. Theinteraction between contact residues from one CH3 domain and contactresidues from a different CH3 domain may for instance be via Van derWaals forces, hydrogen bonds, water-mediated hydrogen bonds, saltbridges or other electrostatic forces, attractive interactions betweenaromatic side chains, disulfide bonds, or other forces known to oneskilled in the art. It was previously shown that approximately one-thirdof the contact amino acid side chains at the human IgG1 CH3 domaininterface can account for the majority of contributions to domainfolding and association. It can further be envisaged that other(neighbouring) amino acid residues may affect the interactions in theprotein-protein interface.

Approaches to interfere with the dimerization of antibody heavy chainshave been employed in the art. Specific engineering in the CH3 domainswas applied in order to favour heterodimerization over homodimerization.Examples of such engineering of the CH3-CH3 interface include theintroduction of complementary protuberance and cavity mutations, alsoknown as ‘knob-into-hole’ approaches as described for instance inWO1998/050431, Ridgeway et al., 1996 and Merchant et al. 1998.

Generally, the method involves introducing a protuberance at theinterface of a first polypeptide and a corresponding cavity in theinterface of a second polypeptide, such that the protuberance can bepositioned in the cavity so as to promote heteromultimer formation andhinder homomultimer formation. “Protuberances” or “knobs” areconstructed by replacing small amino acid side chains from the interfaceof the first polypeptide with larger side chains (e.g. tyrosine ortryptophan). Compensatory “cavities” or “holes” of identical or similarsize to the protuberances are created in the interface of the secondpolypeptide by replacing large amino acid side chains with smaller ones(e.g. alanine or threonine). The protuberance and cavity can be made bysynthetic means such as altering the nucleic acid encoding thepolypeptides or by peptide synthesis.

Using the knob-into-hole technology alone, the proportion of abispecific antibody of interest is at best 87% of the mixture of the 2parental and bispecific antibodies. Merchant et al., succeeded inraising the proportion of bispecific antibodies to 95% of the mixture byintroduction of an additional disulfide bond between the two CH3 domainsin the CH3-CH3 interface. Still, in order to use such bispecificantibody as a medicament, the bispecific antibody has to be purified(separated) from the homodimers and formulated into a pharmaceuticallyacceptable diluent or excipient. Purification of heterodimers from suchmixtures poses a major challenge because of the similarity inphysico-chemical properties of the homodimers and heterodimers. It isone object of the present invention to provide methods for producing abispecific antibody in a single cell clone with a further improvedproportion of the bispecific antibody in the mixture. According to theinvention, knob-into-hole technology can thus be used as one of themeans, alone or together with other means, to achieve said furtherimproved bispecific proportion in a mixture.

Another example of such engineering of the CH3-CH3 interface is providedby a heterodimeric Fc technology that supports the design of bispecificand asymmetric fusion proteins by devising strand-exchange engineereddomain (SEED) CH3 heterodimers. These SEED CH3 heterodimers arederivatives of human IgG and IgA CH3 domains that are composed ofalternating segments of human IgA and IgG CH3 sequences which results inpairs of complementary human SEED CH3 heterodimers, the so-calledSEED-bodies (Davis J H Et al., Protein Engineering, Design & Selection2010(23)195-202; WO2007/110205).

Yet another approach for the production of a given bispecific antibodyof interest is based on electrostatic engineering of contact residueswithin the CH3-CH3 interface that are naturally charged, as for exampledescribed in EP01870459 or US2010/0015133, WO2007/147901, WO2010/129304,Gunasekaran et al (2010) and WO 2009/089004. These publications describemutations in the CH3 domains of heavy chains wherein naturally occurringcharged amino acid contact residues are replaced by amino acid residuesof opposite charge (i.e. a charge reversal strategy). This creates analtered charge polarity across the Fc dimer interface such thatco-expression of electrostatically matched Fc chains support favorableattractive interactions thereby promoting desired Fc heterodimerformation, whereas unfavorable repulsive charge interactions suppressunwanted Fc homodimer formation.

It was described that within the CH3-CH3 interface four unique chargesresidue pairs are involved in the domain-domain interaction. These areD356/K439′, E357/K370′, K392/D399′ and D399/K409′ (numbering accordingto Kabat (1991) where residues in the first chain are separated fromresidues in the second chain by ‘/’ and where the prime (′) indicatesthe residue numbering in the second chain). As the CH3-CH3 interfacedisplays a 2-fold symmetry, each unique charge pair is represented twicein intact IgG (i.e., also K439/D356′, K370/E357′, D3991K392′ andK409/D399′ charge interactions are present in the interface). Takingadvantage of this two-fold symmetry, it was demonstrated that a singlecharge reversion, e.g. K409D in the first chain, or D399′K in the secondchain resulted in diminished homodimer formation due to repulsion ofidentical charges. Combining different charge reversions furtherenhanced this repulsive effect. It was demonstrated that expression ofdifferent CH3 domains comprising different, complementary chargereversions, could drive heterodimerization, resulting in an increasedproportion of the bispecific species in the mixture.

Using the approach described above, it is possible to produce abispecific antibody in a single cell with proportions ranging betweenabout 76% and about 96%. It is an object of the present invention toprovide methods for producing a bispecific antibody in a single cellwith a further improved percentage of desired bispecific antibodies.According to the present invention, electrostatic engineering technologycan be used as one of the means, alone or together with other means,e.g. knob-into-hole approaches, to achieve said further improvedpercentages of desired (bispecific) antibodies.

In one aspect, the present invention provides a method for producing atleast two different Ig-like molecules from a single host cell, whereineach of said two Ig-like molecules comprises two CH3 domains that arecapable of forming an interface, said method comprising providing insaid cell

a) a first nucleic acid molecule encoding a 1^(st) CH3 domain-comprisingpolypeptide chain,

b) a second nucleic acid molecule encoding a 2^(nd) CH3domain-comprising polypeptide chain,

c) a third nucleic acid molecule encoding a 3^(rd) CH3 domain-comprisingpolypeptide chain, and

d) a fourth nucleic acid molecule encoding a 4^(th) CH3domain-comprising polypeptide chain,

wherein at least two of said nucleic acid molecules are provided withmeans for preferential pairing of said 1^(st) and 2^(nd) CH3domain-comprising polypeptides and said 3^(rd) and 4^(th) CH3-domaincomprising polypeptides, said method further comprising the step ofculturing said host cell and allowing for expression of said at leastfour nucleic acid molecules and harvesting said at least two differentIg-like molecules from the culture.

It is often desired to produce more than one (bispecific) antibody, forinstance in order to more efficiently interfere with multiple biologicalpathways involved in a disease process or with the invasion, replicationand/or spreading of a pathogen.

A mixture of more than one bispecific antibody is also particularlyuseful for the treatment of certain diseases. For example, tumor cellsuse many different strategies to develop resistance during treatmentwith antibodies or small molecule drugs. Resistance may involve multiplecell surface receptors and soluble molecules and it is consideredbeneficial to develop antibody-based treatments for cancers that addressmultiple such disease- and escape-associated molecules simultaneously.In case more than 2 such disease- and escape-related target molecules orepitopes are involved, a mixture of bispecific antibodies provides aninnovative and attractive therapeutic format. Preferably, such mixturesof bispecific antibodies are produced by a single cell to facilitate adrug development process that is less complicated from a regulatorypoint of view and cost-effective and feasible from a drug manufacturingand clinical development point of view. In a single cell-based approach,it is desirable to use methods that allow controlled and efficientproduction of the bispecific antibodies, thus reducing or evencompletely abrogating the need of separating the desired mixture ofbispecific IgG molecules from non-desired monospecific IgG molecules. Inthe prior art, mixtures of monospecific and bispecific antibodies havebeen produced by a single cell (WO2004/009618), but these mixturesrepresent complex concoctions of several different bispecific andmonospecific antibody species. It is a further object of the presentinvention to provide means and methods for producing defined mixtures ofbispecific antibodies in single cells. Preferably, methods are providedwhich result in mixtures of (bispecific) antibodies with a proportion ofat least 95%, at least 97% or even more than 99% of dimeric IgGmolecules, irrespective of the amount of monomeric by-products, seeherein below. Typically, in a cell where multiple intact IgG moleculesare produced, half molecules (monomeric by-products) may be present thatcan be simply removed by size exclusion chromatography known in the art.

In one embodiment the present invention provides methods for producing adefined mixture of at least two different Ig-like molecules in singlecells, instead of a single (bispecific) antibody of interest, whereinthe formation of other, undesired dimeric antibody species is diminishedor even absent. The resulting mixture is well defined and itscomposition is controlled by the design of CH3 domain mutants.Furthermore, regulation of expression levels and/or differenttransfection ratios used for expression affects the composition of themixture. In a method according to the invention, a first nucleic acidmolecule encodes a CH3 domain which preferentially pairs with a CH3domain encoded by a second nucleic acid molecule, and a third nucleicacid molecules encodes a CH3 domain which preferentially pairs with aCH3 domain encoded by a fourth nucleic acid molecule. The presentinvention also provides mixtures of at least two different Ig-likemolecules obtainable by the methods of the invention.

As used herein, the term “preferential pairing of said 1^(st) and 2^(nd)CH3 domain-comprising polypeptides” means that essentially all theresulting dimers comprising the 1^(st) CH3 domain-comprising polypeptideand/or the 2^(nd) CH3 domain-comprising polypeptide will be dimersconsisting of one 1^(st) CH3 domain-comprising polypeptide paired withone 2^(nd) CH3 domain-comprising polypeptide. Likewise, the term“preferential pairing of said 3^(rd) and 4^(th) CH3 domain-comprisingpolypeptides” means that essentially all of the resulting dimerscomprising the 3^(rd) CH3 domain-comprising polypeptide and/or the4^(th) CH3 domain-comprising polypeptide will be dimers consisting ofone 3^(rd) CH3 domain-comprising polypeptide paired with one 4^(th) CH3domain-comprising polypeptide. As a result, when nucleic acid moleculesencoding four different (A, B, C, D) CH3 domain-comprising polypeptidesare introduced in a single cell, instead of a mixture of 10 differentIg-like dimers (AA, AB, AC, AD, BB, BC, BD, CC, CD and DD), a mixture ofpredominantly two specific Ig-like molecules is produced.

As explained herein below in more detail, in a preferred embodiment saidfirst CH3-domain comprising polypeptide chain comprises the amino acidsubstitution T366K, and said second CH3-domain comprising polypeptidechain comprises the amino acid substitution L351D. These amino acidchanges are preferred means for preferential pairing of said first andsecond CH3-domain comprising polypeptide chains. Said first CH3-domaincomprising polypeptide chain preferably further comprises the amino acidsubstitution L351K. Moreover, said second CH3-domain comprisingpolypeptide chain preferably further comprises an amino acidsubstitution selected from the group consisting of Y349E, Y349D andL368E, most preferably L368E. In yet another preferred embodiment, saidthird CH3-domain comprising polypeptide chain comprises the amino acidsubstitutions E356K and D399K, and said fourth CH3-domain comprisingpolypeptide chain comprises the amino acid substitutions K392D andK409D.

In a method according to the present invention, each of the CH3-domaincomprising polypeptide chains preferably further comprises a variableregion recognizing a target epitope. The variable regions that are partof the CH3-domain comprising polypeptide chains preferably share acommon light chain. In that case only the VHs of the variable regionsdiffer whereas the VL in all variable regions is essentially the same.Hence, in a preferred aspect a method according to the invention isprovided, which further comprises providing said host cell with anucleic acid molecule encoding a common light chain. In one particularlypreferred embodiment, each of said 4 variable regions of the 4CH3-domain comprising polypeptide chains recognizes a different targetepitope. For instance, if the first nucleic acid molecule encodes aheavy chain that further contains a variable domain with specificity forantigen A, the second nucleic acid molecule encodes a heavy chain thatfurther contains a variable domain with specificity for antigen B, thethird nucleic acid molecule encodes a heavy chain that further containsa variable domain with specificity for antigen C, and the fourth nucleicacid molecule encodes a heavy chain that further contains a variabledomain with specificity for antigen D, a mixture will then be producedcontaining bispecific Ig-like molecules that are specific for AB andbispecific Ig-like molecules that are specific for CD. The formation ofmonospecific antibodies (with AA, BB, CC or DD specificity) orbispecific antibodies with specificity for AC, AD, BC or BD is loweredor even absent due to the means for preferential pairing of said 1^(st)and 2^(nd) CH3 domain-comprising polypeptides and said 3^(rd) and 4^(th)CH3 domain-comprising polypeptides. It is of course, possible to usefurther nucleic acid molecules, for instance encoding a 5^(th) and a6^(th) CH3 domain-comprising polypeptide, in order to produce definedmixtures comprising more than two different Ig-like molecules.

Of note, the ratio of the nucleic acids used in a method according tothe invention does not need to be 1:1:1:1 and the ratio of the resultingIg-like molecules that are expressed does not need to be 1:1. It ispossible to use means known in the art to produce mixtures of antibodieswith optimized ratios. For instance, expression levels of nucleic acidmolecules and hence the ratios of the resulting Ig-like moleculesproduced may be regulated by using different genetic elements such aspromoters, enhancers and repressors or by controlling the genomicintegration site of copy number of the DNA constructs encodingantibodies.

Said means for preferential pairing preferably may comprise engineeredcomplementary knob-into-hole mutations, disulfide bridges, chargemutations including charge reversal mutations, or combinations thereof.The skilled person will appreciate that said means for preferentialpairing may be chosen within a certain type of mutations, i.e. all atleast 4 nucleic acid molecules encoding CH3-domain comprisingpolypeptide chains may for example comprise charge mutations as meansfor preferential pairing. Additionally, also non-engineered wildtype CH3may in certain instances be used for preferential pairing of twowildtype CH3-domain comprising polypeptide chains. In a particularlypreferred embodiment, said means for preferential pairing comprise atleast one CH3 mutation selected from Table B, as explained elsewhere inthis application. One preferred embodiment thus provides a methodaccording to the present invention, wherein all 4 of said nucleic acidmolecules are provided with means for preferential pairing of said1^(st) and 2^(nd) CH3 domain-comprising polypeptides and said 3^(rd) and4^(th) CH3-domain comprising polypeptides, wherein said means forpreferential pairing of said 1^(st) and 2^(nd) CH3 domain-comprisingpolypeptides are different from those means for preferential pairing ofsaid 3^(rd) and 4^(th) CH3-domain comprising polypeptides.

One aspect of the present invention provides a method according to theinvention, wherein said means for preferential pairing of said 1^(st)and 2^(nd) CH3 domain-comprising polypeptides are different from saidmeans for preferential pairing of said 3^(rd) and 4^(th) CH3-domaincomprising polypeptides. By ‘different’ it is meant that the means forpreferential pairing of said 1^(st) and 2^(nd) CH3 domain comprisingpolypeptides are designed such that preferential pairing of the 1^(st)and 2^(nd) chain is favoured. The design is such that essentially nointeraction between the 1^(st) and the 3^(rd) and/or 4^(th) CH3 domaincomprising polypeptide chain will take place. In other words,dimerization between said 1^(st) CH3 domain comprising polypeptide andsaid 3^(rd) or 4^(th) polypeptide is reduced to essentially zero and soforth. The 3^(rd) and the 4^(th) CH3 domain-comprising polypeptides mayeither be wildtype or may comprise means for preferential pairing thatare different from the means for preferential pairing of the 1^(st) and2^(nd) CH3 domains. Current studies have focused on the production of asingle bispecific antibody, using for instance the knob-into-holetechnology or mutations (reversions) of charged contact amino acidspresent in CH3 domains. Production of defined mixtures of at least two(bispecific) Ig-like molecules, without significant co-production ofother dimeric by-products, has, however, not been realized prior to thepresent invention.

The present invention provides methods for the efficient and controlledproduction of a well-defined mixture of Ig-like molecules, with a highproportion of bispecifics in the mixture. Even a proportion of (two)bispecifics of at least 95%, at least 97% or more is obtained in asystem where two bispecifics are desired. This means that only at most5%, at most 3% or less monospecific bivalent by-products are obtained.Of note, the amount of monomeric by-products, i.e. half molecules, isless important since these half-molecules are easily separated fromdimers using their size difference.

In another preferred embodiment, the variable regions of the 1^(st) andthe 2^(nd) CH3-domain comprising polypeptide chains recognize differenttarget epitopes, whereas the variable regions of the 3^(rd) and the4^(th) CH3-domain comprising polypeptide chains recognize the sametarget epitopes. This will result in the predominant production of onekind of bispecific Ig-like molecule and one kind of monospecific Ig-likemolecule. For instance, if the variable regions of the 1^(st) and the2^(nd) CH3-domain comprising polypeptide chains recognize differenttarget epitopes and if the variable regions of the 3^(rd) and the 4^(th)CH3-domain comprising polypeptide chains both recognize the same targetepitope which is different from the target epitopes recognized by the1^(st) and the 2^(nd) CH3-domains, a mixture of Ig-like molecules havingspecificity for AB or CC will be formed. Further provided is therefore amethod according to the invention, wherein the target epitope recognizedby the variable regions of the 3rd and 4^(th) CH3 domain comprisingpolypeptide chain is the same, but different from the target epitoperecognized by the variable region of the or the 2^(nd) CH3-domaincomprising polypeptide chain.

Alternatively, when the variable regions of the 1^(st) and the 2^(nd)CH3-domain comprising polypeptide chains recognize different targetepitopes and when the variable regions of the 3^(rd) and the 4^(th)CH3-domain comprising polypeptide chains both recognize the same epitopeas the 1^(st) or the 2^(nd) CH3-domain comprising polypeptide chains, amixture of Ig-like molecules having specificity for AB and AA, or AB andBB will be formed. A method according to the invention, wherein thetarget epitope recognized by the variable regions of the 3^(rd) and4^(th) CH3 domain comprising polypeptide chain is the same as the targetepitope recognized by the variable region of the 1^(st) or the 2^(nd)CH3-domain comprising polypeptide chain is therefore also herewithprovided.

It is another object of the present invention to provide means andmethods for producing defined mixtures of bispecific antibodies andmonospecific antibodies in a single cell culture. A non-limiting exampleof such well-defined mixture is a mixture of bispecific antibodies withspecificity AB and monospecific antibodies with specificity AA. Anotherexample is a mixture of bispecific antibodies with specificity AB andmonospecific antibodies with specificity BB. Yet another example is amixture of bispecific antibodies with specificity AB and monospecificantibodies with specificity CC. Again, preferably means and methods areprovided which yield mixtures of antibodies of interest with at least90%, more preferably at least 95% and most preferably at least 97% oreven more than 99% of desired antibodies.

In yet another embodiment, a method according to the invention isprovided wherein the variable regions of the 1^(st) and the 2^(nd)CH3-domain comprising polypeptide chains recognize the same targetepitope, whereas the variable regions of the 3^(rd) and the 4^(th)CH3-domain comprising polypeptide chains recognize a second targetepitope which differs from the target epitope recognized by said 1^(st)and 2^(nd) variable regions. This will result in the predominantproduction of monospecific Ig-like molecules having either specificityfor AA or specificity for BB. The formation of bispecific Ig-likemolecules is diminished or even avoided. In several embodiments it ispreferred to produce mixtures of monospecific antibodies in a singlecell, rather than mixtures of bispecific antibodies. For instance whencross-linking of two identical target molecules is desired, or when twotargets are located too far away from each other so that they cannot bebound by a single bispecific antibody. It can also be advantageous toproduce mixtures of monospecific antibodies in a single cell as themixture can be regarded as a single therapeutic product. In the art, thetherapeutic efficacy and safety of various monospecific antibodies hasalready been proven and market authorisation has been obtained.Production of mixtures of monospecific antibodies in a single cell willthus facilitate the testing for efficacy and safety of several of suchmixtures and will reduce the efforts and costs for regulatory approvaland manufacturing. There are, however, currently no methods availablefor producing specific mixtures of monospecific antibodies in a singlecell wherein the formation of bispecific by-products is reduced to below5%. It is another object of the present invention to provide means andmethods for producing such well-defined homodimeric antibody mixtures insingle cells wherein the formation of bispecific antibodies is reducedto below 5%.

Hence, a method according to the present invention is suitable for theproduction of any desired mixture of bispecific and/or monospecificIg-like molecules. Again, it is possible to use further nucleic acidmolecules, for instance encoding a 5^(th) and a 6^(th) (and 7^(th) and8^(th) and so forth) CH3 domain-comprising polypeptide, in order toproduce defined mixtures comprising more than two different Ig-likemolecules.

Preferably, in a method according to the present invention at least twoCH3 domains are used that comprise at least one combination of mutationsprovided by the present invention. Through these mutations novelspecific interactions are formed between two CH3 domains. Thesemutations according to the present invention are discussed below in moredetail.

The term ‘Ig-like molecule’ as used herein means a proteinaceousmolecule that possesses at least one immunoglobulin (Ig) domain. SaidIg-like molecule comprises a sequence comprising the function of atleast an immunoglobulin CH3 domain, preferably the sequence comprises anIgG1 CH3 domain. Proteinaceous molecules that possess at least a CH3domain can be further equipped with specific binding moieties. The CH3domains of the present invention, containing means for preferentialpairing, can thus be used for preferential pairing of two CH3-domaincomprising proteinaceous molecules to design desired heterodimericbinding molecules or mixtures of binding molecules. Binding moietiesthat can be engineered to the CH3-domain comprising proteinaceousmolecules can be any binding agent, including, but not limited to,single chain Fvs, single chain or Tandem diabodies (TandAb®), VHHs,Anticalins®, Nanobodies®, a BiTE®, a Fab, ankyrin repeat proteins orDARPINs®, Avimers®, a DART, a TCR-like antibody, Adnectins®, Affilins®,Trans-Bodies®, Affibodies®, a TrimerX®, MicroProteins, Fynomers®,Centyrins® or a KALBITOR®. In a preferred embodiment, the bindingmoieties are antibody variable regions (i.e. VH/VL combinations).Variable regions that are part of the CH3-domain comprising polypeptidechains preferably share a common light chain. In that case, only the VHsof the variable regions differ whereas the VL in all variable regions isessentially the same.

Alternatively, or in addition, other molecules can be engineered to theCH3 domains of the present invention, including cytokines, hormones,soluble ligands, receptors and/or peptides.

In a more preferred embodiment, said Ig-like molecule comprises a fulllength Fc backbone. In a most preferred embodiment, the Ig-likemolecules are antibodies. The variable regions of these antibodiespreferably share a common light chain, but they may differ in their VHregions. The term ‘antibody’ as used herein means a proteinaceousmolecule belonging to the immunoglobulin class of proteins, containingone or more domains that bind epitope on an antigen, where such domainsare derived from or share sequence homology with the variable region ofan antibody. Antibodies are known in the art and include severalisotypes, such as IgG1, IgG2, IgG3, IgG4, IgA, IgE, and IgM. An antibodyaccording to the invention may be any of these isotypes, or a functionalderivative and/or fragment of these. In a preferred embodiment, Ig-likemolecules are produced that are antibodies of the IgG isotype becauseIgG antibodies e.g. have a longer half life as compared to antibodies ofother isotypes.

Antibodies produced with methods according to the present invention canhave sequences of any origin, including murine and human sequences.Antibodies can consist of sequences from one origin only, such as fullyhuman antibodies, or they can have sequences of more than one origin,resulting for instance in chimeric or humanized antibodies. Antibodiesfor therapeutic use are preferably as close to natural antibodies of thesubject to be treated as possible (for instance human antibodies forhuman subjects). Antibody binding can be expressed in terms ofspecificity and affinity. The specificity determines which antigen orepitope thereof is bound by the binding domain. The affinity is ameasure for the strength of binding to a particular antigen or epitope.Specific binding is defined as binding with affinities (K_(D)) of atleast 1×10⁻⁵ M, more preferably 1×10⁻⁷ M, more preferably higher than1×10⁻⁹M. Typically, monoclonal antibodies for therapeutic applicationshave affinities of up to 1×10⁻¹⁰ M or even higher. The term ‘antigen’ asused herein means a substance or molecule that, when introduced into thebody, triggers the production of an antibody by the immune system. Anantigen, among others, may be derived from pathogenic organisms, tumorcells or other aberrant cells, from haptens, or even from selfstructures. At the molecular level, an antigen is characterized by itsability to be bound by the antigen-binding site of an antibody. Alsomixtures of antigens can be regarded as ‘antigen’, i.e. the skilledperson would appreciate that sometimes a lysate of tumor cells, or viralparticles may be indicated as ‘antigen’ whereas such tumor cell lysateor viral particle preparation exists of many antigenic determinants. Anantigen comprises at least one, but often more, epitopes. The term‘epitope’ as used herein means a part of an antigen that is recognizedby the immune system, specifically by antibodies, B cells, or T cells.Although epitopes are usually thought to be derived from non-selfproteins, sequences derived from the host that can be recognized arealso classified as epitopes.

The term ‘CH3 domain’ is well known in the art. The IgG structure hasfour chains, two light and two heavy chains; each light chain has twodomains, the variable and the constant light chain (VL and CL) and eachheavy chain has four domains, the variable heavy chain (VH) and threeconstant heavy chain domains (CH1, CH2, CH3). The CH2 and CH3 domainregion of the heavy chain is called Fc (Fragment crystallizable)portion, Fc fragment, Fc backbone or simply Fc. The IgG molecule is aheterotetramer having two heavy chains that are held together bydisulfide bonds (—S—S—) at, the hinge region and two light chains. Theheavy chains dimerize through interactions at the CH3-CH3 domaininterface and through interactions at the hinge region. The number ofhinge disulfide bonds varies among the immunoglobulin subclasses(Papadea and Check 1989). The Fc fragment of an immunoglobulin moleculeis a dimer of the two C-terminal constant regions, i.e. CH2 and CH3domains, of the heavy chain. Among its physiological functions areinteractions with the complement system and with specific receptors onthe surface of a variety of cells. Interactions between the CH3 domainsof two individual heavy chains are known to play an important role indriving heavy chain dimerization. Thus, CH3 domains direct theassociation of antibody heavy chains, and it is known that the interfacebetween CH3 domains contains more than 20 contact residues from eachchain that play a role in the CH3-CH3 interaction (Deisenhofer J.,Biochemistry 1981(20)2361-2370; Miller S., J. Mol. Biol.1990(216)965-973; Padlan, Advances in Protein Chemistry 1996 (49)57-133). The CH3 variants of the present invention can thus be used inassociation with other antibody domains to generate full lengthantibodies that are either bispecific monospecific. The specificity ofthe antibody as defined by the VH/VL combinations typically does notaffect the heavy chain dimerization behaviour that is driven by the CH3domains.

The terms ‘contact residue’, ‘contact amino acid’, ‘interface residue’and ‘interface amino acid’ as used herein typically refers to any aminoacid residue present in the CH3 domain that can be involved ininterdomain contacts, as can be calculated by technologies known in theart, including calculating solvent accessible surface area (ASA) of theCH3 domain residues in the presence and absence of the second chain (Leeand Richards J. Mol. Biol. 1971(55)379) where residues that showdifference (>1 Å²) in ASA between the two calculations are identified ascontact residues. Contact residues that have been identified includeresidues at positions 347, 349, 350, 351, 352, 353, 354, 355, 356, 357,360, 364, 366, 368, 370, 390, 392, 394, 395, 397, 399, <100, 405, 407,409, 439 according to the EU numbering system (Table A).

TABLE A List of CH3 domain interface residues Interface residueContacting residues in in chain A chain B Q347 K360 Y349 S354, D356,E357, K360 T350 S354, R355 L351 L351, P352, P353, S354, T366 S354 Y349,T350, L351 R355 T350 D356 Y349, K439 E357 Y349, K370 K360 Q347, Y349S364 L368, K370 T366 L351, Y407 L368 S364, K409 K370 E357, S364 N390S400 K392 L398, D399, S400, F405 T394 T394, V397, F405, Y407 P395 V397V397 T394, P395 D399 K392, K409 S400 N390, K392 F405 K392, T394, K409Y407 T366, T394, Y407, K409 K409 L368, D399, F405, Y407 K439 D356Contact residues within the CH3-CH3 interface can either be amino acidsthat are charged, or amino acid residues that are neutral. The term‘charged amino acid residue’ or ‘charged residue’ as used herein meansamino acid residues with electrically charged side chains. These caneither be positively charged side chains, such as present in arginine(Arg, histidine (His, H) and lysine (Lys, K) or can be negativelycharged side chains, such as present in aspartic acid (Asp, D) andglutamic acid (Glu, E). The term ‘neutral amino acid residue’ or neutralresidue as used herein refers to all other amino acids that do not carryelectrically charged side chains. These neutral residues include serine(Ser, S), threonine (Thr, T), asparagine (Asn, N), glutamine (GLu, Q),Cysteine (Cys, C), glycine (Gly, G), proline (Pro, P), alanine (Ala, A),valine (Val, V), isoleucine (Ile, I), leucine (Leu, L), methionine (Met,M), phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, T).

The term ‘CH3-CH3 domain interface’, or ‘CH3 interface’, ‘CH3-CH3pairing’, ‘domain interface’ or simply ‘interface’, as used herein,refers to the association between two CH3 domains of separate CH3-domaincomprising polypeptides that is a result of interacting amino acidresidues, i.e. at least one interaction between an amino acid of a firstCH3 domain and an amino acid of a second CH3 domain. Such interaction isfor instance via Van der Waals forces, hydrogen bonds, water-mediatedhydrogen bonds, salt bridges or other electrostatic forces, attractiveinteractions between aromatic side chains, the formation of disulfidebonds, or other forces known to one skilled in the art.

As used herein, said means for preferential pairing of the first andsecond CH3 domain-comprising polypeptides and said third and fourth CH3domain-comprising polypeptide can be any means known in the art. In oneembodiment, at least one nucleic acid molecule encodes a CH3 domainwhich contains at a contact residue position a large amino acid residue(i.e. a “knob” or “protuberance”) such as for instance H, F, Y, W, I orL, whereas at least one other nucleic acid molecule encodes a CH3 domainwhich contains at a complementary contact residue position a small aminoacid residue (i.e. a “hole” or “cavity”) such as for instance G, A, S, Tor V. The resulting CH3 domains will preferentially pair with each otherdue to the steric conformation of said contact amino acids. Theknob-into-hole technology is described herein before in more detail. Ina further embodiment of the present invention, at least one nucleic acidmolecule encodes a CH3 domain which contains at a contact residueposition that is naturally charged, i.e. a naturally occurring K, H, H,D or E, an amino acid that now carries the opposite charge as comparedto wildtype, whereas at least one other nucleic acid molecule encodes aCH3 domain which contains at a complementary contact residue positionthat is naturally charged, an amino acid that now carries the oppositecharge as compared to wildtype. The resulting engineered CH3 domainswill preferentially pair with each other due to the opposite charges ofsaid contact amino acids, whereas pairing of identical CH3 domains willbe diminished due to electrostatic repulsion. In one embodiment, CH3mutations as described in EP01870459, WO 2009/089004, Gunasekaran et al(2010), are used. In one embodiment, the means for preferential pairingof said 1^(st) and 2^(nd) CH3 domain-comprising polypeptides are “knob”and “hole” amino acid residues and the means for preferential pairing ofsaid 3^(th) and 4^(th) CH3 domain-comprising polypeptides arecharge-engineered amino acids. Preferably, both said means forpreferential pairing of said 1^(st) and 2^(nd) CH3 domain-comprisingpolypeptides and said 3^(th) and 4^(th) CH3 domain-comprisingpolypeptides are charge-engineered amino acids. In one embodiment,different amino acid residues are engineered for preferential pairing ofsaid 1^(st) and 2^(nd) CH3 domain-comprising polypeptides as compared tothe amino acid residues that are engineered for preferential pairing ofsaid 3^(th) and 4^(th) CH3 domain-comprising polypeptides. In aparticularly preferred embodiment at least a first and a second nucleicacid molecule encode CH3 domains with novel mutations as provided by thepresent invention. As described herein below in more detail, the presentinvention provides novel CH3 mutations which enable the production ofcertain bispecific Ig-like molecules of interest without a significantamount of undesired (dimeric) by-products. The present invention alsoprovides novel CH3 mutations which enable the production of certainmonospecific Ig-like molecules of interest without a significant amountof undesired (dimeric) by-products. The use of at least one of these CH3mutations according to the present invention is therefore, preferred.

The term ‘polypeptide’, ‘polypeptide molecule’ or ‘polypeptide chain’ asused herein refers to a chain of amino acids that are covalently joinedtogether through peptide bonds. Proteins are typically made up of one ormore polypeptide molecules. One end of every polypeptide, called theamino terminal or N-terminal, has a free amino group. The other end,with its free carboxyl group, is called the carboxyl terminal orC-terminal. Polypeptides according to the present invention may havegone through post-translational modification processes and may e.g. beglycosylated. The CH3 domain-comprising polypeptide chains of thepresent invention thus refer to polypeptide chains that at leastencompass an Ig CH3 domain and that may have gone throughpost-translational modification processes.

The term “nucleic acid molecule” as used herein is defined as a moleculecomprising a chain of nucleotides, more preferably DNA and/or RNA. Inone embodiment, double-stranded RNA is used. In other embodiments anucleic acid molecule of the invention comprises other kinds of nucleicacid structures such as for instance a DNA/RNA helix, peptide nucleicacid (PNA), locked nucleic acid (LNA) and/or ribozyme. Hence, the term“nucleic acid molecule” also encompasses a chain comprising non-naturalnucleotides, modified nucleotides and/or non-nucleotide building blockswhich exhibit the same function as natural nucleotides.

The present invention further provides a method for making a host cellfor production of at least two different Ig-like molecules, the methodcomprising the step of introducing into said host cell nucleic acidsequences encoding at least a first, a second, a third and a fourthCH3-domain comprising polypeptide chain, wherein at least two of saidnucleic acid sequences are provided with means for preferential pairingof said first and second CH3-domain comprising polypeptides and saidthird and fourth CH3-domain comprising polypeptides, wherein saidnucleic acid sequences are introduced consecutively or concomitantly.

It is a further aspect of the present invention to provide a method formaking a host cell for production of a heterodimeric Ig-like molecule,the method comprising the step of introducing into said host cellnucleic acid sequences encoding at least a first and a second CH3-domaincomprising polypeptide chain, wherein said first CH3 domain-comprisingpolypeptide chain comprises at least one substitution of a neutral aminoacid residue by a positively charged amino acid residue and wherein saidsecond CH3 domain-comprising polypeptide chain comprises at least onesubstitution of a neutral amino acid residue by a negatively chargedamino acid residue, wherein said nucleic acid sequences are introducedconsecutively or concomitantly. Said methods for making said host cellspreferably further comprise the step of introducing into said host cella nucleic acid sequence encoding a common light chain.

Also provided herein is a recombinant host cell comprising nucleic acidsequences encoding at least a first, a second, a third and a fourthCH3-domain comprising polypeptide chain, wherein at least two of saidnucleic acid molecules are provided with means for preferential pairingof said first and second CH3-domain comprising polypeptides and saidthird and fourth CH3-domain comprising polypeptides. The inventionfurthermore provides a recombinant host cell comprising nucleic acidsequences encoding at least a first and a second CH3-domain comprisingpolypeptide chain, wherein said first CH3 domain-comprising polypeptidechain comprises at least one substitution of a neutral amino acidresidue by a positively charged amino acid residue and wherein saidsecond CH3 domain-comprising polypeptide chain comprises at least onesubstitution of a neutral amino acid residue by a negatively chargedamino acid residue.

A recombinant host cell according to the invention preferably furthercomprises a nucleic acid sequence encoding a common light chain.

A “host cell” according to the invention may be any host cell capable ofexpressing recombinant DNA molecules, including bacteria such as forinstance. Escherichia (e.g. E. coli), Enterobacter, Salmonella,Bacillus, Pseudomonas, Streptomyces, yeasts such as S. cerevisiae, K.lactis, P. pastoris, Candida, or Yarrowia, filamentous fungi such asNeurospora, Aspergillus oryzae, Aspergillus nidulans and Aspergillusniger, insect cells such as Spodoptera frugiperda SF-9 or SF-21 cells,and preferably mammalian cells such as Chinese hamster ovary (CHO)cells, BHK cells, mouse cells including SP2/0 cells and NS-0 myelomacells, primate cells such as COS and Vero cells, MDCK cells, BRL 3Acells, hybridomas, tumor-cells, immortalized primary cells, human cellssuch as W138, HepG2, HEK293, HT1080 or embryonic retina cells such asPER. C6, and the like. Often, the expression system of choice willinvolve a mammalian cell expression vector and host so that theantibodies can be appropriately glycosylated. A human cell line,preferably PER.C6, can advantageously be used to obtain antibodies witha completely human glycosylation pattern. The conditions for growing ormultiplying cells (see e, g. Tissue Culture, Academic Press, Kruse andPaterson, editors (1973)) and the conditions for expression of therecombinant product may differ somewhat, and optimization of the processis usually performed to increase the product proportions and/or growthof the cells with respect to each other, according to methods generallyknown to the person skilled in the art. In general, principles,protocols, and practical techniques for maximizing the productivity ofmammalian cell cultures can be found in Mammalian Cell Biotechnology: aPractical. Approach (M, Butler, ed., IRL Press, 1991). Expression ofantibodies in recombinant host cells has been extensively described inthe art (see e.g. EP0120694; EP0314161; EP0481790; EP0523949; U.S. Pat.No. 4,816,667; WO 00/63403). The nucleic acid molecules encoding thelight and heavy chains may be present as extrachromosomal copies and/orstably integrated into the chromosome of the host cell, the latter ispreferred.

It is a further aspect of the present invention to provide a culture ofrecombinant host cells according to the invention, or a culture ofrecombinant host cells obtainable or obtained by a method according tothe invention, said culture either producing at least two differentIg-like molecules or a heterodimeric Ig-like molecule.

To obtain expression of nucleic acid sequences encoding the CH3domain-comprising polypeptides, it is well known to those skilled in theart that sequences capable of driving such expression can befunctionally linked to the nucleic acid sequences encoding the CH3domain-comprising polypeptides. Functionally linked is meant to describethat the nucleic acid sequences encoding the CH3 domain-comprisingpolypeptides or precursors thereof is linked to the sequences capable ofdriving expression such that these sequences can drive expression of theCH3 domain-comprising polypeptides or precursors thereof. Usefulexpression vectors are available in the art, e.g. the pcDNA vectorseries of Invitrogen. Where the sequence encoding the polypeptide ofinterest is properly inserted with reference to sequences governing thetranscription and translation of the encoded polypeptide, the resultingexpression cassette is useful to produce the polypeptide of interest,referred to as expression. Sequences driving expression may includepromoters, enhancers and the like, and combinations thereof. Theseshould be capable of functioning in the host cell, thereby drivingexpression of the nucleic acid sequences that are functionally linked tothem. Promoters can be constitutive or regulated, and can be obtainedfrom various sources, including viruses, prokaryotic, or eukaryoticsources, or artificially designed. Expression of nucleic acids ofinterest may be from the natural promoter or derivative thereof or froman entirely heterologous promoter. Some well-known and much usedpromoters for expression in eukaryotic cells comprise promoters derivedfrom viruses, such as adenovirus, e.g. the E1A promoter, promotersderived from cytomegalovirus (CMV), such as the CMV immediate early (IE)promoter, promoters derived from Simian Virus 40 (SV40), and the like.Suitable promoters can also be derived from eukaryotic cells, such asmethallothionein (MT) promoters, elongation factor 1α (EF-1α) promoter,actin promoter, an immunoglobulin promoter, heat shock promoters, andthe like. Any promoter or enhancer/promoter capable of drivingexpression of the sequence of interest in the host cell is suitable inthe invention. In one embodiment the sequence capable of drivingexpression comprises a region from a CMV promoter, preferably the regioncomprising nucleotides −735 to +95 of the CMV immediate early geneenhancer/promoter. The skilled artisan will be aware that the expressionsequences used in the invention may suitably be combined with elementsthat can stabilize or enhance expression, such as insulators, matrixattachment regions, STAR elements (WO 03/004704), and the like. This mayenhance the stability and/or levels of expression.

Protein production in recombinant host cells has been extensivelydescribed, e.g. in Current Protocols in Protein Science, 1995, Coligan JE, Dunn B M, Ploegh H L, Speicher D W, Wingfield P T, ISBN0-471-11184-8; Bendig, 1988. Culturing a cell is done to enable it tometabolize, and/or grow and/or divide and/or produce recombinantproteins of interest. This can be accomplished by methods well known topersons skilled in the art, and includes but is not limited to providingnutrients for the cell. The methods comprise growth adhering tosurfaces, growth in suspension, or combinations thereof. Severalculturing conditions can be optimized by methods well known in the artto optimize protein production yields. Culturing can be done forinstance in dishes, roller bottles or in bioreactors, using batch,fed-batch, continuous systems, hollow fiber, and the like. In order toachieve large scale (continuous) production of recombinant proteinsthrough cell culture it is preferred in the art to have cells capable ofgrowing in suspension, and it is preferred to have cells capable ofbeing cultured in the absence of animal- or human-derived serum oranimal- or human-derived serum components. Thus purification is easierand safety is enhanced due to the absence of additional animal or humanproteins derived from the culture medium, while the system is also veryreliable as synthetic media are the best in reproducibility.

Ig-like molecules are expressed in host cells and are harvested from thecells or, preferably, from the cell culture medium by methods that aregenerally known to the person skilled in the art. After harvesting,these Ig-like molecules may be purified by using methods known in theart. Such methods may include precipitation, centrifugation, filtration,size-exclusion chromatography, affinity chromatography, cation- and/oranion-exchange chromatography, hydrophobic interaction chromatography,and the like. For a mixture of antibodies comprising IgG molecules,protein A or protein G affinity chromatography can be suitably used (seee.g. U.S. Pat. Nos. 4,801,687 and 5,151,504).

Ig-like molecules, and/or mixtures thereof, produced with methodsaccording to the present invention preferably have a common light chain.Further provided is, therefore, a method according to the invention,further comprising providing said host cell with a nucleic acid moleculeencoding a common light chair. This is a light chain that is capable ofpairing with at least two different heavy chains, thereby formingfunctional antigen binding domains. A functional antigen binding domainis capable of specifically binding to an antigen. Preferably, a commonlight chain is used that is capable of pairing with all heavy chainsproduced with a method according to the invention, thereby formingfunctional antigen binding domains, so that mispairing of unmatchedheavy and light chains is avoided. In one aspect, only common lightchains with one identical amino acid sequence are used. Alternatively,those of skill in the art will recognize that “common” also refers tofunctional equivalents of the light chain of which the amino acidsequence is rot identical. Many variants of said light chain existwherein mutations (deletions, substitutions, additions) are present thatdo not materially influence the formation of functional binding regions.Such variants are thus also capable of binding different heavy chainsand forming functional antigen binding domains. The term ‘common lightchain’ as used herein thus refers to light chains which may be identicalor have some amino acid sequence differences while retaining the bindingspecificity of the resulting antibody after pairing with a heavy chain.It is for instance possible to prepare or find light chains that are notidentical but still functionally equivalent, e.g. by introducing andtesting conservative amino acid changes, and/or changes of amino acidsin regions that do not or only partly contribute to binding specificitywhen paired with the heavy chain, and the like. A combination of acertain common light chain and such functionally equivalent variants isencompassed within the term “common light chain”. Reference is made toWO 2004/009618 for a detailed description of the use of common lightchains. Preferably, a common light chain is used in the presentinvention which is a germline-like light chain, more preferably agermline light chain, preferably a rearranged germline human kappa lightchain, most preferably either the rearranged germline human kappa lightchain IgV_(K)1-39/J_(K) or IGV_(K)3-20/J_(K).

Alternatively, the skilled person may select, as an alternative to usinga common light chain and to avoid mispairing of unmatched heavy andlight chains, means for forced pairing of the heavy and light chain,such as for example described in WO2009/080251 WO2009/080252 and/orWO2009/080253.

The present invention provides novel engineered CH3 domains as well asnovel combinations of CH3 mutations. Before the present invention,charged contact amino acids of CH3 domains that, were known to beinvolved in CH3-CH3 pairing were substituted by amino acids of oppositecharge (charge reversal), thereby influencing the CH3-CH3 pairing. Themutations according to the present invention are an inventivealternative to this approach, because now CH3 amino acids that arenon-charged or neutral in wildtype CH3 are substituted with chargedresidues. The present invention in this embodiment does not exchangecharged contact amino acids by amino acids of opposite charge butsubstitutes non-charged CH3 amino acids for charged ones. The approachof the present invention provides not only a method for efficientlysteering the dimerization of CH3 domains but also has the advantage thatat least one additional charge-charge interaction in the CH3 interfaceis created, in view of this additional charge-charge interaction on topof the existing charge-pairs in the CH3-CH3 interface, the dimersaccording to the invention are generally more stable as compared to thewild type dimers (the wild type dimer is defined as a bispecific IgG(AB) without CH3 engineering in contrast to its parental homodimers (AAor BB)). Moreover, it has surprisingly become possible to increase theproportion of one or more Ig-like molecules of interest in a mixtureeven further. As described herein before, methods known in the art forpreferential production of a bispecific antibody typically involves theproduction of some undesired dimeric side products. For instance, theproportion of a bispecific antibody of interest using the knob-into-holetechnology is at best 87%, whereas the electrostatic engineeringapproach wherein charged contact amino acids are substituted by aminoacids of opposite charge, also results in proportions of up to 96% (seefor instance Example 11). Quite surprisingly, the present inventors havesucceeded in introducing mutations that further enhance the proportionof an Ig-like molecule of interest in a mixture. For instance, Example17 discloses a method using mutations according to the presentinvention, wherein the proportion of a bispecific antibody of interestwas raised to such extent that no dimeric by-product was detectable inthe resulting mixture at all. Unpaired half-molecules consisting of onlya single heavy chain paired with a common light chain were present tosome extent in the mixtures, but these are the result of unbalancedexpression of the heavy chains and can be easily separated from themixture by size exclusion chromatography. Hence, with such mutationsaccording, to the present invention, a bispecific Ig-like molecule canbe produced in a single cell with a high proportion with essentially nocontaminating dimeric by-products being present, which is particularlysuitable for the production of a pharmaceutical composition.

One preferred embodiment of the present invention therefore provides amethod for producing a heterodimeric Ig-like molecule from a singlecell, wherein said Ig-like molecule comprises two CH3 domains that arecapable of forming an interface, said method comprising providing insaid cell

a. A first nucleic acid molecule encoding a 1^(st) CH3 domain-comprisingpolypeptide chain,

b. A second nucleic acid molecule encoding a 2^(nd) CH3domain-comprising polypeptide chain,

wherein said first CH3 domain-comprising polypeptide chain comprises atleast one substitution of a neutral amino acid residue by a positivelycharged amino acid residue and wherein said second CH3 domain-comprisingpolypeptide chain comprises at least one substitution of a neutral aminoacid residue by a negatively charged amino acid residue, said methodfurther comprising the step of culturing said host cell and allowing forexpression of said two nucleic acid molecules and harvesting saidheterodimeric Ig-like molecule from the culture.

Said method preferably further comprises the step of providing said hostcell with a nucleic acid molecule encoding a common light chain, whichhas advantages as outlined herein before.

The amino acids at position 366 of one CH3 domain and position 351 of asecond CH3 domain have been reported to be a pair of contact residues inthe CH3-CH3 interface, meaning that they are located sufficiently closeto each other in the three-dimensional conformation of the resultingIg-like molecule in order to be capable of interacting with each other.Hence, the first CH3 domain will preferentially pair with the second CH3domain.

In one embodiment, threonine (T) at position 366 of a first CH3 domainis replaced by a first charged amino acid and leucine (L) at position351 of a second CH3 domain is replaced by a second charged amino acid,wherein said first and second charged amino acids are of oppositecharge. If the first CH3 domain-comprising polypeptide, that carries acharged residue at position 366, further comprises a variable domainwhich has specificity for antigen A, and if the second CH3domain-comprising polypeptide, that carries an oppositely chargedresidue at position 351, further comprises a variable domain which hasspecificity for antigen B, bispecific Ig-like molecules with an ABspecificity will be predominantly formed. Further provided is thereforea method according to the present invention, wherein said means forpreferential pairing of said 1^(st) and 2^(nd) CH3 domain-comprisingpolypeptides or said means for preferential pairing of said 3^(rd) and4^(th) CH3 domain-comprising polypeptides are a substitution ofthreonine at position 366 of said 1^(st) or 3^(rd) CH3 domain by a firstcharged amino acid and substitution of leucine at position 351 of said2^(nd) or 4^(th) CH3 domain by a second charged amino acid, wherein saidfirst and second charged amino acids are of opposite charge.

One preferred combination of mutations according to the presentinvention is the substitution of threonine (T) by lysine (K) at position366 of a first CH3 domain-comprising polypeptide which further comprisesa variable domain (for instance with specificity A) and the substitutionof leucine (L) by aspartic acid (D) at position 351 of a second CH3domain-comprising polypeptide which further comprises a variable domain(for instance with specificity B). This is denoted as a T366K/L351′Dpair mutation. As explained before, the amino acids at position 366 ofone CH3 domain and position 351 of a second CH3 domain have beenreported to be a pair of contact residues in the CH3-CH3 interface. Thelysine that is introduced at position 366 and the aspartic acidintroduced at position 351 have opposite charges, so that these aminoacids will electrostatically attract each other. Hence, the first CH3domain will preferentially attract the second CH3 domain and Ig-likemolecules comprising a first CH3 domain containing lysine at position366 paired with a second CH3 domain containing aspartic acid at position351 will be predominantly formed. If the first CH3-domain-comprisingpolypeptide has specificity for antigen A, and if the second CH3domain-comprising polypeptide has specificity for antigen B, bispecificIg-like molecules with ‘AB’ specificity will be predominantly formed.Nota bone, in some embodiments the specificity of the variable domainsof both said first and second CH3-domain comprising polypeptide chainsmay be the same, which will result in the formation of monospecificIg-like molecules (for instance with ‘AA’ specificity). As mentionedabove, one of the advantages of the mutations according to the presentinvention is the fact that a novel interaction between a newlyintroduced pair of charged amino acids is created, instead of replacingexisting charged amino acid interactions. This was not previouslydisclosed or suggested. One aspect of the invention therefore provides amethod according to the present invention for producing at least twodifferent Ig-like molecules from a single host, cell, wherein said1^(st) CH3 domain-comprising polypeptide chain comprises the amino acidsubstitution T366K, and said 2^(nd) CH3 domain-comprising polypeptidechain comprises the amino acid substitution L351D. One embodimentprovides a method for producing a heterodimeric Ig-like molecule from asingle cell, wherein said Ig-like molecule comprises two CH3 domainsthat are capable of forming an interface, said method comprisingproviding in said cell:

-   -   a first nucleic acid molecule encoding a 1^(st) CH3        domain-comprising polypeptide chain, and    -   a second nucleic acid molecule encoding a 2^(nd) CH3        domain-comprising polypeptide chain,        wherein said first CH3 domain-comprising polypeptide chain        comprises the amino acid substitution T366K and wherein said        second CH3 domain comprising polypeptide chain comprises the        amino acid substitution L351D, said method further comprising        the step of culturing said host cell and allowing for expression        of said two nucleic acid molecules and harvesting said        heterodimeric Ig-like molecule from the culture.

Using the above mentioned amino acid substitutions according to thepresent invention, it has become possible to produce a heterodimericIg-like molecule from a single cell, whereby the presence ofcontaminating homodimers is less than 5%, preferably less than 2%, morepreferably less than 1%, or, most preferably, whereby contaminatinghomodimers are essentially absent. One embodiment therefore provides amethod for producing a heterodimeric Ig-like molecule from a singlecell, wherein said. Ig-like molecule comprises two CH3 domains that arecapable of forming an interface and wherein the presence ofcontaminating homodimers is less than 5%, preferably less than 2%, morepreferably less than 1%, and most preferably contaminating homodimersare essentially absent, said method comprising providing in said cell:

-   -   a first nucleic acid molecule encoding 1^(st) CH3        domain-comprising polypeptide chain, and    -   a second nucleic acid molecule encoding a 2^(nd) CH3        domain-comprising polypeptide chain,        wherein said first CH3 domain-comprising polypeptide chain        comprises the amino acid substitution T366K and wherein said        second CH3 domain comprising polypeptide chain comprises the        amino acid substitution L351D, said method further comprising        the step of culturing said host cell and allowing for expression        of said at two nucleic acid molecules and harvesting said        heterodimeric Ig-like molecule from the culture.

Preferably, a method according to the present invention for producing atleast two different Ig-like molecules, or a method according to theinvention for producing a heterodimeric Ig-like molecule, is providedwherein said first CH3-domain comprising polypeptide chain furthercomprises the amino acid substitution L351K. It is further preferredthat said second CH3-domain comprising polypeptide chain furthercomprises an amino acid substitution selected from the group consistingof Y349E, Y349D and L368E. Most preferably said second CH3-domaincomprising polypeptide chain further comprises the amino acidsubstitution L368E.

Thus, in a preferred embodiment the above mentioned T366K/L351′Dmutations according to the present invention are further combined withthe substitution of leucine (L) by glutamic acid (E) at position 368 ofthe second CH3 domain. This is, for example, denoted as aT366K/L351′D,L368′E mutation (but alternative ways of denoting are alsopossible, such as T336K/L351D-L368E T366K/L351D,L368E T366KL351D,L368E). As shown in Example 17, introduction of this mutationaccording to the invention into a first CH3 domain-comprisingpolypeptide with specificity for antigen A, and a second CH3domain-comprising polypeptide with specificity for antigen B results ina particular good proportion of bispecific Ig-like molecules with dualAB specificity. With this mutational pair it has even become possible toobtain bispecific antibody without detectable amount of homodimersformed. A particularly preferred embodiment therefore provides a methodfor producing a heterodimeric Ig-like molecule from a single cell,wherein said Ig-like molecule comprises two CH3 domains that are capableof forming an interface and wherein the presence of contaminatinghomodimers is less than 5%, preferably less than 2%, more preferablyless than 1%, and most preferably contaminating homodimers areessentially absent, said method comprising providing in said cell:

-   -   a first nucleic acid molecule encoding a 1^(st) CH3        domain-comprising polypeptide chain, and    -   a second nucleic acid molecule encoding a 2^(nd) CH3        domain-comprising polypeptide chain,        wherein said first CH3 domain-comprising polypeptide chain        comprises the amino acid substitution T366K and wherein said        second CH3 domain comprising polypeptide chain comprises the        amino acid substitutions L351D and L368E, said method further        comprising the step of culturing said host cell and allowing for        expression of said at two nucleic acid molecules and harvesting        said heterodimeric Ig-like molecule from the culture.

In yet another preferred embodiment, threonine (T) is substituted bylysine (K) at position 366 of a first CH3 domain and leucine (L) issubstituted by aspartic acid (D) at position 351 of a second CH3 domainand tyrosine (Y) is substituted by glutamic acid (E) at position 349 ofsaid second CH3 domain. This is for example denoted as aT366K/L351′D,Y349′E mutation but other ways of denoting these mutationsmay include for example T366K-L351D:Y349E, or T366K/L351D,Y349E orsimply T366K/L351DY349E. Residue Y349 is a neighboring residue of theresidue at position 351 that may contribute to dimer interactions.According to in silico data, Y349E adds to the stability of theheterodimer (lower in silico scores) as well as to the destabilizationof the monodimer (higher in silico scores) and glutamic acid (E)position 349 is more favorable than aspartic acid (D). Thus,introduction of a second amino acid substitution in the second CH3domain comprising polypeptide, comprising already the amino acidsubstitution at position 351, favors heterodimerization further.

A particularly preferred embodiment therefore provides a method forproducing a heterodimeric Ig-like molecule from a single cell, whereinsaid Ig-like molecule comprises two CH3 domains that are capable offorming an interface and wherein contaminating homodimers are less than5%, more preferably less than 2%, even more preferably less than 1%, andmost preferably essentially absent, said method comprising providing insaid cell:

-   -   a first nucleic acid molecule encoding a 1^(st) CH3        domain-comprising polypeptide chain, and    -   a second nucleic acid molecule encoding a 2^(nd) CH3        domain-comprising polypeptide chain,        wherein said first CH3 domain-comprising polypeptide chain        comprises the amino acid substitution T366K and wherein said        second CH3 domain comprising polypeptide chain comprises the        amino acid substitutions L351D and Y349E, said method further        comprising the step of culturing said host cell and allowing for        expression of said at two nucleic acid molecules and harvesting        said heterodimeric Ig-like molecule from the culture.

In yet another preferred embodiment, threonine (T) is substituted bylysine (K) position 366 of a first CH3 domain and leucine (L) issubstituted by aspartic acid (D) at position 351 of a second CH3 domainand tyrosine (1) is substituted by glutamic acid (E) at position 349 ofsaid second CH3 domain and leucine (1) is substituted by glutamic acid(E) at position 368 of said second CH3 domain. This is denoted as aT366K/L351′D,Y349′E,L368′E mutation. The two residues Y349 and L368 areresidues that may contribute to dimer interactions. According to the insilico data, Y349E and L368E add to the stability of the heterodimer(lower in silico scores) as well as to the destabilization of the BBdimer (higher in silico scores) and glutamic acids (E) on positions 349and 368 are more favorable than aspartic acids (D). Thus, introductionof a second and third amino acid substitution in the B-chain, whichalready comprises the amino acid substitution at position 351, favorsheterodimerization further. A particularly preferred embodimenttherefore provides a method for producing a heterodimeric Ig-likemolecule from a single cell, wherein said Ig-like molecule comprises twoCH3 domains that are capable of forming an interface and whereincontaminating homodimers are less than 5%, more preferably less than 2%,even more preferably less than 1%, and most preferably essentiallyabsent, said method comprising providing in said cell:

-   -   a first nucleic acid molecule encoding a 1^(st) CH3        domain-comprising polypeptide chain, and    -   a second nucleic acid molecule encoding a 2^(nd) CH3        domain-comprising polypeptide chain,        wherein said first CH3 domain-comprising polypeptide chain        comprises the amino acid substitution T366K and wherein said        second CH3 domain comprising polypeptide chain comprises the        amino acid substitutions L351D and Y349E and L368E, said method        further comprising the step of culturing said host cell and        allowing for expression of said at two nucleic acid molecules        and harvesting said heterodimeric Ig-like molecule from the        culture.

In yet another preferred embodiment, threonine (T) is substituted bylysine (K) at Position 366 of a first CH3 domain and leucine (L) issubstituted by lysine (K) at position 351 of said first CH3 domain andleucine (L) is substituted by aspartic acid (D) at position 351 of asecond CH3 domain and leucine (L) is substituted by glutamic acid (E) atposition 368 of said second CH3 domain. This is denoted as aT366K,L351K/L351′D,L368′E mutation. This mutation also enhances theproportion of the (bispecific) antibody of interest, as shown in theExamples. Also with this mutation it has become possible to obtainbispecific antibody without any detectable amount of homodimers formed.Further provided is therefore a method for producing a heterodimericIg-like molecule from a single cell, wherein said Ig-like moleculecomprises two CH3 domains that are capable of forming an interface andwherein contaminating homodimers are less than 5%, preferably less than2%, more preferably less than 1%, and most preferably essentiallyabsent, said method comprising providing in said cell:

-   -   a first nucleic acid molecule encoding a 1^(st) CH3        domain-comprising polypeptide chain, and    -   a second nucleic acid molecule encoding a 2^(nd) CH3        domain-comprising polypeptide chain,        wherein said first CH3 domain-comprising polypeptide chain        comprises the amino acid substitutions T366K and L351K, and        wherein said second CH3 domain comprising polypeptide chain        comprises the amino acid substitutions L3510 and L368E, said        method further comprising the step of culturing said host cell        and allowing for expression of said at two nucleic acid        molecules and harvesting said heterodimeric Ig-like molecule        from the culture.

In yet another preferred embodiment, threonine (T) is substituted bylysine (K) at position 366 of a first CH3 domain and leucine (L) issubstituted by lysine (K) at position 351 of said first CH3 domain andleucine (L) is substituted by aspartic acid (D) at position 351 of asecond CH3 domain and tyrosine (Y) is substituted by aspartic acid (D)at position 349 of said second CH3 domain and arginine (R) issubstituted by aspartic acid (D) at position 355 of said second CH3domain. This is denoted as a T366K,L351K/L351′D,Y349′D,R355′D mutation.The T366K-L351K/L351′D-Y349′D pair may be further improved by the R355′Dmutation in the B-chain, which results in a higher BB-in silico score,but also the AB in silico score is slightly higher. Further provided istherefore a method for producing a heterodimeric Ig-like molecule from asingle cell, wherein said Ig-like molecule comprises two CH3 domainsthat are capable of forming an interface and wherein contaminatinghomodimers are less than 5%, more preferably less than 2%, even morepreferably less than 1%, and most preferably essentially absent, saidmethod comprising providing in said cell:

-   -   a first nucleic acid molecule encoding a 1^(st) CH3        domain-comprising polypeptide chain, and    -   a second nucleic acid molecule encoding a 2^(nd) CH3        domain-comprising polypeptide chain,        wherein said first CH3 domain-comprising polypeptide chain        comprises the amino acid substitutions T366K and L351K, and        wherein said second CH3 domain comprising polypeptide chain        comprises the amino acid substitutions L351D and Y349D and        R355D, said method further comprising the step of culturing said        host cell and allowing for expression of said at two nucleic        acid molecules and harvesting said heterodimeric Ig-like        molecule from the culture.

Table B provides an overview of mutations that can be introduced in CH3domains as preferred means for preferential pairing to create eitherheterodimers or homodimers.

TABLE B AA substitutions in CH3 Construct # Preferentially pairs with -(wildtype) — Wildtype E356K, D399K 1 Construct 2 or 3 K392D, K409D 2Construct 1 K392D, K409D, K439D 3 Construct 1 K392D, D399K, K409D 4Construct 4 E356K, E357K, K439D, K370D 5 Construct 5 T366W 6 Construct 7T366S, L368A, Y407V 7 Construct 6 T366K 43 Construct 63, 69, 70, 71, 73L351D 63 Construct 43, 68 T366K, L351K 68 Construct 63, 69, 70, 71, 72,75 L351D, L368E 69 Construct 43, 68 L351E, Y349E 70 Construct 43, 68L351D, Y349E 71 Construct 43, 68 L351D, R355D 72 Construct 43, 68 L351D,Y349E, L368E 73 Construct 43 L351D, Y349D, R355D 75 Construct 68

A method according to the present invention for producing at least twodifferent Ig-like molecules, or a method according to the invention forproducing a heterodimeric Ig-like molecule, wherein said means forpreferential pairing of said 1^(st) and 2^(nd) CH3 domain-comprisingpolypeptides and/or said means for preferential pairing of said 3^(rd)and 4^(th) CH3 domain-comprising polypeptides comprise at least onecombination of mutations as depicted in Table B is therefore alsoprovided herewith. Preferably, said means for preferential pairing ofsaid 1^(st) and 2^(nd) CH3 domain-comprising polypeptides and said meansfor preferential pairing of said 3^(rd) and 4^(th) CH3 domain-comprisingpolypeptides comprise at least two combinations of mutations as depictedin Table B.

The present invention also provides novel combinations of CH3 mutationswith which it has become possible to produce a mixture of at least twomonospecific molecules in a single cell, wherein contaminatingbispecific Ig-like molecules are less than 5%, preferably more than 2%,even more preferably less than 1%, and most preferably even essentiallyabsent. These mutations according to the invention are, therefore,particularly suitable for the production of a mixture of monospecificantibodies, which is for instance advantageous when a high level ofcrosslinking of two identical target molecules is desired, when thedensity of antibodies on a target cells needs to be high enough torecruit certain effector functions such as complement-mediated lysis ofa tumor cell, or when two targets are located too far away from eachorder so that they cannot be bound by as single bispecific antibody, orin order to simplify regulatory approval procedures. In such cases, itis often desired to optimize the production platform for suchmonospecific antibodies. As shown in Example 10, the present inventionprovides the insight that when lysine (K) at position 392 of a first CH3domain-comprising polypeptide (for instance having specificity A) issubstituted by aspartic acid (D) and when aspartic acid (D) at position399 of said first CH3 domain-comprising polypeptide is substituted bylysine (K) and when lysine (K) at position 409 of said first CH3domain-comprising polypeptide is substituted by aspartic acid (D), ithas become possible to produce a mixture of at least two differentmonospecific Ig-like molecules in a single cell, including monospecificIg like molecules with specificity AA, wherein the formation ofbispecific by-products (bispecific Ig-like molecules) is reduced tobelow 5%, or even to below 3%, or even essentially not detectable atall. Hence, the above mentioned combination of mutations (denoted hereinas K392D, D399K, K409D) is particularly preferred for the production ofa mixture of monospecific Ig-like molecules. The skilled person willappreciate that functional variants thereof, i.e., K392E, D399R, K409E,may result in similar effects. Additionally, double mutants comprisingD399K and K409D substitutions, or other functional variants such as e.g.K392D and K409D, D399R and K409E and so forth, may also result insimilar effects.

The same holds true for a combination of mutations wherein glutamic acid(E) at position 356 of a first CH3 domain-comprising polypeptide issubstituted by lysine (K) and wherein glutamic acid (E) at position 357of said first CH3 domain-comprising polypeptide is substituted by lysine(K) and wherein lysine (K) at position 439 of said first CH3domain-comprising polypeptide is substituted by aspartic acid (D) andwherein lysine (K) at position 370 of said first CH3 domain-comprisingpolypeptide is substituted by aspartic acid (D). This combination ofmutations (denoted herein as E356K, E357K, K439D, K370D) is alsoparticularly preferred for the production of a mixture of monospecificIg-like molecules. The skilled person will appreciate that functionalvariants thereof, i.e., E356R, E357R, K439E, K370E, may result insimilar effects. Additionally, triple or double mutants comprising E356Kand K439D, and E357K and K370D substitutions, or other functionalvariants may also result in similar effects. A further embodimenttherefore provides a method for producing at least two differentmonospecific Ig-like molecules from a single host cell, wherein each ofsaid two Ig-like molecules comprises two CH3 domains that are capable offorming an interface, said method comprising providing in said cell

a) a first nucleic acid molecule encoding a 1^(st) CH3 domain-comprisingpolypeptide chain having a specificity A,

b) a second nucleic acid molecule encoding a 2^(nd) CH3domain-comprising polypeptide chain having a specificity B,

wherein said first CH3 domain-comprising polypeptide chain comprises aK392D, D399K, K409D mutation and said second CH3 domain-comprisingpolypeptide chain comprises either a wildtype CH3 domain or comprises aE356K, E357K, K439D, K370D mutation, said method further comprising thestep of culturing said host cell and allowing for expression of saidnucleic acid molecules and harvesting said at least two differentIg-like molecules from the culture.

An alternative embodiment provides a method for producing at least twodifferent monospecific Ig-like molecules from a single host cell,wherein each of said two Ig-like molecules comprises two CH3 domainsthat are capable of forming an interface, said method comprisingproviding in said cell

a) a first nucleic acid molecule encoding a 1^(st) CH3 domain-comprisingpolypeptide chain having a specificity A,

b) a second nucleic acid molecule encoding a 2^(nd) CH3domain-comprising polypeptide chain having a specificity B,

wherein said first CH3 domain-comprising polypeptide chain compriseseither a wildtype CH3 domain or comprises a K392D, D399K, K409D mutationand said second CH3 domain-comprising polypeptide chain comprises aE356K, E357K, K439P, K370D mutation, said method further comprising thestep of culturing said host cell and allowing for expression of saidnucleic acid molecules and harvesting said at least two differentIg-like molecules from the culture.

As shown in Example 10, two monospecific Ig-like molecules can beproduced in a single cell, wherein the formation of bispecific Ig-likemolecules is essentially undetectable. The skilled person may select a3^(rd) nucleic acid molecule encoding a wildtype or engineered CH3domain-comprising polypeptide chain to provide to said host cell suchthat a mixture of 3 monospecific antibodies is produced, and so forth.

In one aspect of the invention, a method according to the invention forproducing at least two different Ig-like molecules or for producing aheterodimeric Ig-like molecule is provided wherein each of theCH3-domain comprising polypeptide chains further comprises a variableregion recognizing a different target epitope, wherein the targetepitopes are located on the same molecule. This often allows for moreefficient counteraction of the (biological) function of said targetmolecule as compared to a situation wherein only one epitope istargeted. For example, a heterodimeric Ig-like molecule maysimultaneously bind to 2 epitopes present on, e.g., growth factorreceptors or soluble molecules critical for tumors cells to proliferate,thereby effectively blocking several independent signalling pathwaysleading to uncontrolled proliferation, and any combination of at leasttwo Ig-like molecules may simultaneously bind to 2, or even 3 or 4epitopes present on such growth factor receptors or soluble molecules.

In a preferred embodiment, the target molecule is a soluble molecule. Inanother preferred embodiment, the target molecule is a membrane-boundmolecule.

In another aspect of the invention, a method according to the inventionfor producing at least two different Ig-like molecules or for producinga heterodimeric Ig-like molecule is provided wherein each of theCH3-domain comprising polypeptide chains further comprises a variableregion recognizing a target epitope, wherein the target epitopes arelocated on different molecules. In this case, each of the differenttarget molecules may either be a soluble molecule or a membrane-boundmolecule. In one embodiment, the different target molecules are solublemolecules. Alternatively, one target molecule is a soluble moleculewhereas the second target molecule is a membrane bound molecule. In yetanother alternative, both target molecules are membrane bound molecules.In one embodiment the different target molecules are expressed on thesame cells, whereas in other embodiments the different target moleculesare expressed on different cells. As a non-limiting example, anyheterodimeric Ig-like molecule or any combination of at least twoIg-like molecules may be suitable for simultaneously blocking multiplemembrane-bound receptors, neutralizing multiple soluble molecules suchas cytokines or growth factors for tumor cells or for neutralizingdifferent viral serotypes or viral strains.

One preferred embodiment provides a method according to the inventionfor producing at least two different Ig-like molecules or for producinga heterodimeric Ig-like molecule, wherein at least one of said targetepitopes is located on a tumor cell. Alternatively, or additionally, atleast one of said target epitopes is located on the surface of aneffector cell. This is for instance suitable for recruitment of T cellsor NK cells for tumor cell killing. For instance, at least one Ig-likemolecule is produced with a method according to the invention that iscapable of recruiting immune effector cells, preferably human immuneeffector cells, by specifically binding to a target molecule located onimmune effector cells. In a further embodiment, said immune effectorcell is activated upon binding of the Ig-like molecule to the targetmolecule. Recruitment of effector mechanisms may for instance encompassthe redirection of immune modulated cytotoxicity by administering anIg-like molecule produced by a method according to the invention that iscapable of binding to a cytotoxic trigger molecule such as the T cellreceptor or an Fc gamma receptor, thereby activating downstream immuneeffector pathways. The term ‘immune effector cell’ or ‘effector cell’ asused herein refers to a cell within the natural repertoire of cells inthe mammalian immune system which can be activated to affect theviability of a target cell. Immune effector cells include cells of thelymphoid lineage such as natural killer (NK) cells, T cells includingcytotoxic T cells, or B cells, but also cells of the myeloid lineage canbe regarded as immune effector cells, such as monocytes or macrophages,dendritic cells and neutrophilic granulocytes. Hence, said effector cellis preferably an NK cell, a r cell, a B cell, a monocyte, a macrophage,a dendritic cell or a neutrophilic granulocyte.

Target antigens present on immune effector cells may include CD3, CD16,CD25, CD28, CD64, CD89, NKG2D and NKp46. Further provided is therefore amethod according to the invention for producing at least two differentIg-like molecules or for producing a heterodimeric Ig-like molecule,wherein said target epitope is located on a CD3, CD16, CD25, CD28, CD64,CD89, NKG2D or a NKp46 molecule.

The viability of a target cell may include cell survival, proliferationand/or ability to interact with other cells.

In one aspect the present invention thus provides methods according tothe invention for producing a heterodimeric Ig-like molecule, whereineach of the CH3-domain comprising polypeptide chains further comprises avariable region recognizing a target epitope. In one embodiment, each ofthe 2 variable regions of the CH3-domain comprising polypeptide chainsrecognizes the same target epitope but with different affinities. Inanother embodiment, each of the 2 variable regions of the CH3-domaincomprising polypeptide chains recognizes a different target epitope. Inanother embodiment, the different target epitopes are located on thesame target molecule, which can be either a membrane-bound molecule or asoluble molecule. In another embodiment, the different target epitopesare located on different target molecules, which can be either expressedon the same cells or on different cells. Alternatively, the differenttarget molecules can be soluble molecules, or one target molecule can bea soluble molecule whereas the second target molecule is a membranebound molecule. In a preferred embodiment, at least one of the targetmolecules of the heterodimeric Ig-like molecule is located on a tumorcell. In yet another preferred embodiment, at least one of the targetmolecules of the heterodimeric Ig-like molecule is located on aneffector cell (i.e. an NK cell, a T cell, a B cell, a monocyte, amacrophage, a dendritic cell or a neutrophilic granulocyte, and saidtarget epitope may be located on a CD3, CD16, CD25, CD28, CD64, CD89,NKG2D or a NKp46 molecule).

In a preferred embodiment, a method according to the invention forproducing at least two different Ig-like molecules or for producing aheterodimeric Ig-like molecule is provided, wherein said at least twodifferent Ig-like molecules are antibodies, most preferably antibodiesof the IgG isotype, even more preferably the IgG1 isotype, as describedherein above.

Further provided is an Ig-like molecule, a heterodimeric molecule or amixture of at least two Ig-like molecules, obtainable by a methodaccording to the present invention. Said (heterodimeric) Ig-likemolecule or mixture of Ig-like molecules preferably comprises at leastone CH3 mutation as depicted in Table B. An (heterodimeric) Ig-likemolecule or a mixture of at least two Ig-like molecules, comprising atleast one mutation as depicted in Table B is therefore also herewithprovided, as well as a pharmaceutical composition comprising at leastone Ig-like molecule, or a mixture of at least two Ig-like molecules,according to the present invention. In one embodiment said Ig-likemolecule is a bispecific Ig-like molecule, such as a bispecificantibody. In another embodiment said Ig-like molecule is a monospecificIg-like molecule, such as a monospecific antibody. One preferredembodiment provides a mixture of at least two different Ig-likemolecules obtainable by a method according to the invention, whereinsaid at least two different. Ig-like molecules bind to differentepitopes on the same antigen and/or to different epitopes on different,antigens. Further provided is a heterodimeric Ig-like moleculeobtainable by a method according to the invention, wherein saidheterodimeric Ig-like molecule binds to different epitopes on the sameantigen and/or to different epitopes on different antigens. Advantagesand preferred uses of such mixtures and antibodies are described hereinbefore. The invention also provides a mixture of at least two different.Ig-like molecules obtainable by a method according to the invention,wherein said at least two different Ig-like molecules comprise at leastone heterodimeric Ig like molecule. In one embodiment, two of said atleast two different. Ig-like molecules are heterodimeric Ig-likemolecules. Yet another preferred embodiment provides a heterodimericantibody comprising two CH3 domains, wherein one of said two CH3 domainscomprises the amino acid substitutions L351D and L368E and wherein theother of said two CH3 domains comprises the amino acid substitutionsT366K and L351K. These amino acid substitutions are preferred means forpreferential pairing of said two CH3 domains, as explained before. Theamino acid substitutions L351D and L368E in one of said two CH3 domainsand the amino acid substitutions T366K and L351K in the other of saidtwo CH3 domains are together dubbed the ‘DEKK combination of mutations’,‘DEKK variant’, ‘DEKK pair’, ‘DEKK engineered CH3 domains’, ‘DEKK’ oralternative names referring to DEKK are used. The CH3 domain thatcarries the amino acid substitutions L351D and L368E is also dubbed‘DE-side’ and the CH3 domain that carries the amino acid substitutionsT366K and L351K is also dubbed ‘the KK-side’.

Also provided is a pharmaceutical composition comprising a(heterodimeric) Ig-like molecule, or a mixture of at least two Ig-likemolecules obtainable by any method according to the invention. Said(heterodimeric) Ig-like molecule, or said at least two Ig-like moleculesaccording to the invention is/are preferably (an) antibody/antibodies.Said pharmaceutical composition may comprise said (heterodimeric)Ig-like molecule, a mixture comprising monospecific bispecific Ig-likemolecules, or a combination of monospecific and bispecific Ig-likemolecules. In addition, a pharmaceutical composition according to theinvention comprises a pharmaceutically acceptable carrier. As usedherein, such ‘pharmaceutically acceptable carrier’ includes any and allsolvents, salts, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible. Depending on the route ofadministration (e.g., intravenously, subcutaneously, intra-articularlyand the like) the Ig-like molecules may be coated in a material toprotect the Ig-like molecules from the action of acids and other naturalconditions that may inactivate the Ig-like molecules. In one aspect, apharmaceutical composition comprising a mixture of at least two Ig-likemolecules obtainable by any method according to the invention isprovided, wherein said at least two different. Ig-like molecules havebeen produced by recombinant host cells according to the presentinvention. Furthermore, a pharmaceutical composition is providedcomprising a heterodimeric Ig-like molecule obtainable by any methodaccording to the invention, wherein said heterodimeric Ig-like moleculehas been produced by recombinant host cells according to the presentinvention.

A nucleic acid molecule encoding a CH3 domain-comprising polypeptidechain that comprises at least one mutation as depicted in Table B isalso provided herewith, as well as a recombinant host cell comprising atleast, one nucleic acid molecule encoding a CH3 domain-comprisingpolypeptide chain that comprises at least one mutation as depicted inTable B.

The invention is further illustrated by the following examples. Theseexamples are not limiting the invention in any way, but merely serve toclarify the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A) schematic representation of construct vector MV1057. Thestuffer region is the region into which an antibody VH region is cloned.B) schematic representation of phage display vector MV1043.

FIG. 2: amino acid sequence of wildtype IgG1Fc (SEQ ID NO: 1), aspresent in construct vector MV1057 (EU numbering scheme applied).

FIG. 3: nucleotide and amino acid sequences (SEQ ID NOS: 2-7) of VHregions used for cloning into the various constructs.

FIG. 4: mass spec data of transfections A, G and H.

FIG. 5: mass spec data of transfections M and U.

FIG. 6: mass spec data of transfection O.

FIG. 7: prevention of homodimerisation by substitution of neutral aminoacids for charged amino acids.

FIG. 8: Native MS spectrum of transfection sample ZO (T366K/L351′D) (A)and Convoluted MS spectrum of transfection sample ZO (T366K/L351′D). Thesecond/main peak represents the bispecific molecule (B).

FIG. 9: HADDOCK scores on experimentally verified mutation pairs

FIG. 10: Cartoons of interactions in the CH3-CH3 interface; A)K409D:K392D/D399′K:E356′K, B) D399K:E356K/D399′K:E356′K, C)K409D:K392D/K409′D:K392′D

FIG. 11: HADDOCK scores for various 366/351′ charge mutants

FIG. 12: Cartoons of interactions in the CH3-CH3 interface; A)L351D/L351′D, B) L351D:S354A;R355D/L351′D:S354A:R355′D

FIG. 13: HADDOCK scores for additional charge mutations around positionL351

FIG. 14: HADDOCK scores for additional charge mutations around positionT366 in chain A and position L351 in chain B.

FIG. 15: Cartoons of interactions in the CH3-CH3 interface

FIG. 16: HADDOCK scores for variants around T366/L351

FIG. 17: HADDOCK scores for additional variants around T366/L35

FIG. 18: Examples of nMS spectra for bispecific IgG obtained after theco-expression of construct T366K,L351K with either construct L351D (lefthand panel) or L351D,Y349E (right hand panel), zoomed in on a singlecharge state of the full IgG (half bodies not shown)

FIG. 19: A) Results of native MS showing relative abundances of AA, AB,BB, A and B (total of all species is 100%); B) idem but now without ABto have a better overview on the undesired species AA, BB, A and B

FIG. 20: Results of thermostability assay. Squares: wildtype; triangles:charge reversal pair E356K:D399K/K392D:K409D; circles: mutant CH3combinations as indicated above each graph.

FIG. 21: Results of 10× freeze-thaw experiment. 1122=1^(st) parentalantibody BB; 1337=2^(nd) parental antibody AA; wildtype=AA, AB, BB;CR=bispecific of charge reversal pair E356K:D399K/K392D:K409D; 3-6 and9-12=bispecific molecules from combinations 3-6 and 9-12 from Table 15.

FIG. 22: Results in serum stability, measured by ELISA using fibrinogenas coated antigen. A) ELISA data with IgG samples diluted to 0.5 μg/ml;B) ELISA data with IgG samples diluted to 0.05 μg/ml; Results arenormalized to the T=0 days time point (100%). 1337=2^(nd) parentalantibody AA; wildtype=AA, AB, BB; CR=bispecific of charge reversal pairE356K:D399K/K392D:K409D; 3-6 and 9-12=bispecific molecules fromcombinations 3-6 and 9-12 from Table 15.

FIG. 23: nMS results of ratio experiments with transfection ratios from1:5 to 5:1. A) DEKK combination of mutations, with specificity ‘A’ onthe DE-side and ‘B’ on the KK-side; B) DEKK combination of mutations,with specificity ‘C’ on the DE-side and ‘B’ on the KK-side; C) chargereversal combination of mutations, with specificity ‘A’ on theE356K:D399K-side and ‘B’ on the K392D:K409D-side

FIG. 24: nMS results of transfections #1-11 from Table 20.

FIG. 25: HADDOCK scores for dimers with different CH3 engineeredvectors. Grey bars: Desired species AB and CD; black bars: undesiredspecies AA, BB, CC, DD, AC, BC, AD, BD.

FIG. 26: SDS-PAGE of transfections #1-11 from Table 20. Control samplesDE/KK, DE/DE and KK/KK are also included.

FIG. 27: nMS transfections #9 (A) and #11 (B).

FIG. 28: nMS of gel filtrated samples 1516:1516 (A), 1337:1337 (B) and1516:1337 (C).

FIG. 29: serum levels of samples of DEKK engineered antibody and its twoparental antibodies (pK study).

EXAMPLES Example 1 Amino Acid Substitutions to Create Various DifferentCH3-Domains

In order to have a wide variety of Ig-like molecules that differ intheir CH3 domains such that pairing of CH3-domain comprising Ig-likemolecules is preferentially promoted or inhibited, a number of aminoacid substitutions that were known to promote heterodimer formation, aswell as a number of alternative amino acid substitutions that were notpreviously reported nor tested but that were chosen to promote homodimerformation, were introduced into a construct vector (construct vectorMV1057; FIG. 1A). The construct vector MV1057 comprises nucleic acidsequences encoding the normal wildtype IgG1 Fc part, as depicted in FIG.2. Table 1 lists the amino acid substitutions that were introduced inthis wildtype Fc, resulting in a series of seven constructs. Allconstructs were made at Geneart. Constructs 1, 2 and 3, or alternativesthereof, have previously been described to drive heterodimerization(EP01870459, WO2009/089004) as have constructs 6 and 7 (WO98/50431).Constructs 4 and 5 are new and are designed to promote homodimerization.

TABLE 1 % bispecific product AA substitutions in CH3 construct # Willpair with reported - (wildtype) — - (wildtype)  ~50% E356K, D399K 1Construct 2 or 3 ~100% K392D, K409D 2 Construct 1 ~100% K392D, K409D,K439D 3 Construct 1 ~100% K392D, D399K, K409D 4 Construct 4 E356K,E357K, K439D, 5 Construct 5 K370D T366W 6 Construct 7 ~86.7%  T366S,L368A, Y407V 7 Construct 6 ~86.7% 

Example 2 Cloning of VII into Constructs with CH3 Mutations

Several antibody VII regions with known specificities and known abilityto pair with the human IGKV1-39 light chain were used for cloning intothese constructs.

As indicated earlier, all CH3 variants can be used in association withother antibody domains to generate full length antibodies that areeither bispecific or monospecific. The specificity of the antibody asdefined by the VH/VL combinations will not affect the heavy chaindimerization behaviour that is driven by the CH3 domains. Model VH/VLcombinations were used throughout the studies, wherein all Nits arebased on the germline human IGKV1-39 and VHs vary. FIG. 3 provides fullsequences and specificities of the antibody VH regions used throughoutthe studies. The MF coding refers to internal Merus designation forvarious VHs, e.g. VH MF1337 has specificity for tetanus toxoid, MF1025for porcine thyroglobulin, MF1122 for bovine fibrinogen. VH regionspresent in phage display vector MV1043 (FIG. 1B) are digested withrestriction enzymes SfiI and BstEII (New England Biolabs/cat#R0123L andR0162L/according to manufacturer's instructions) that release the VHfragment from this vector. Vector MV1057 is digested with SfiI andBstEII according to standard procedures (according to manufacturer'sinstructions). Fragments and vector are purified over gel(Promega/cat#V3125/according to manufacturer's instructions) to isolatethe cut vector and VH gene inserts. Both are combined by ligation afterwhich the ligation is transformed into E. coli DH5α(Invitrogen/cat#12297-016/according to manufacturer's instructions).After overnight selection single colonies are picked and vectors with acorrect insert identified by sequencing.

Example 3 Transfection and Expression of Full IgG in HEK293T Cells

Transfection of the various plasmids encoding the recloned VH variants,and further encoding the common light chain huIGKV1-39, in HEK293T cellswas performed according to standard procedures such that IgG couldexpress (de Kruif et al Biotech Bioeng. 2010). After transfection, IgGexpression levels in supernatants were measured using the ForteBIOOctet-QK system, which is based on Bio-Layer Interferometry (BLI) andwhich enables real-time quantitation and kinetic characterization ofbiomolecular interactions; for details see www.fortebio.com. Whenexpression levels exceeding 5 μg/ml were measured, the IgG was purifiedusing Protein A affinity purification.

Example 4 Purification of IgG

Culture supernatants were purified using protein A columns (GEHealthcare/cat#11-0034-95/according to manufacturer's instructions) andeluted in 0.1 M citrate buffer pH 3.0 and immediately neutralized in anequal volume of 1.0 M Tris-HCL pH 8.0 or directly rebuffered to PBSusing a desalting column. Alternatively one could purify IgG usingprotein A beads (sepharose beads CL-4B, GE healthcare cat #170780-01)

Example 5 Ag-Specific ELISA's

Antigen specific ELISAs were performed to establish binding activityagainst the antigens and capture ELISAs were carried out to demonstratebinding activity of the bispecific antibodies. Biotinylated secondantigen was used for detection of the complex. (de Kruif et al BiotechBioeng. 2010)

Example 6 SDS-PAGE

The purified IgG mixtures were analysed by SDS-PAGE (NuPAGE® 4-12%bis-tris gel/Invitrogen/cat#NP0323BOX) under reduced and non-reducingconditions according to standard procedures, and staining of proteins ingel was carried out with colloidal blue (PageBlue™ protein stainingsolution/Fermentas/cat#RO571).

Example 7 Enzymatic Deglycosylation of IgG1

As there is heterogeneity in the glycosylation of the IgGs, the proteinswere deglycosylated in order to create a single product with a distinctmass, suitable for mass spectrometric analysis. One unit ofN-glycosidase (PNGase F; Roche Diagnostics, Mannheim, Germany) wasincubated per 10 μg of IgG1, overnight at 37° C. Buffer exchange using10 kDa MWCO centrifugal filter columns (Millipore) was performed toremove the original purification buffer (0.1 M citrate buffer pH 3.0/1.0M Tris-HCL pH 8.0) and to rebutter to PBS. Similar buffer exchangeprocedures were performed to remove the detached glycan chains, and tochange the buffer to 150 mM ammonium acetate pH 7.5. Filters were washedwith 200 μl 150 mM ammonium acetate pH 7.5, for 12 min 11,000 rpm and 4°C. After washing 50 μl deglycosylated IgG was loaded on the filter and450 μl of 150 mM ammonium acetate pH 7.5 was added, subsequentlyfollowed by another centrifugation round of 12 min at 11,000 rpm at 4°C. In total the centrifugation was repeated 5 tunes, each time fresh 150mM ammonium acetate pH 7.5 buffer was added to a total volume of 500 μl.After the last centrifugation step the remaining buffer exchangeddeglycosylated IgG1, approximately 25 μl, was collected and transferredto an eppendorf tube, ready for mass spectrometric analysis.

Example 8 Native Mass Spectrometric Analysis

Mass Spectrometry was used to identify the different IgG species in thepurified IgG mixtures and to establish in what ratios these IgG speciesare present. Briefly, 2-3 μl at a 1 μM concentration in 150 mM ammoniumacetate pH 7.5 of IgG's were loaded into gold-plated borosilicatecapillaries made in-house (using a Sutter P-97 puller [Sutterinstruments Co., Novato, Calif., USA] and an Edwards Scancoat sixsputter-coater [Edwards Laboratories, Milpitas, Calif., USA]) foranalysis on a LCT 1 mass spectrometer (Waters Corp., Milford, Mass.,USA), adjusted for optimal performance in high mass detection (Tahallahet al., RCM 2001). A capillary voltage of 1300 V was used and a samplingcone voltage of 200 V; however, these settings were adjusted when ahigher resolution of the ‘signal-to-noise’ ratio was required. Thesource backing pressure was elevated in order to promote collisionalcooling to approximately 7.5 mbar. To measure the IgG1's underdenaturing conditions the proteins were sprayed at a 1 μM concentrationin 5% formic acid.

Example 9 Data Processing and Quantification

Processing of the acquired spectra was performed using MassLynx 4.1software (Waters Corp Milford, Mass., USA). Minimal smoothing was used,after which the spectra were centered. The mass of the species wascalculated using each charge state in a series. The correspondingintensities of each charge state were assigned by MassLynx and summed.This approach allowed the relative quantification of all species in asample. Alternatively, quantification of the peaks can be performedusing area-under-the-curve (AUC) methods, known in the art. All analyseswere repeated three times to calculate standard deviations of both themasses of the IgG's as well as their relative abundance.

Example 10 Mixtures of 2 or 3 Monospecific Antibodies from a Single Cell

Several antibody VH regions with known specificities and known abilityto pair with the human IGKV1-39 light chain (FIG. 3)) were used forreckoning into the wildtype construct vector MV1067, or in construct 4or construct 5 of Table 1, resulting in vectors I-III (Table 2). Theresulting vectors I, II and III, each containing nucleic acid sequencesencoding for the common human light chain as well as an Ig heavy chainwith different CH3 region and different VH specificity, weresubsequently transfected into cells, either alone to demonstrateformation of intact monospecific antibodies only, or in combination withone or two other construct vectors to obtain mixtures of twomonospecific or three monospecific antibodies. Table 3 depicts thetransfection schedule and results.

TABLE 2 VH specificity inserted in different constructs VH VH Antigenmass Merus Cloned in Vector gene specificity (Da) designation construct# I IGHV Tetanus (A) 13703 MF1337 wildtype 1.08 II IGHV Thyroglobulin12472 MF1025 4 3.23 (B) III IGHV Fibrinogen 12794 MF1122 5 3.30 (C)

TABLE 3 transfection schedule and results # different Transfec- Experi-AA BB CC Other mono-specifics Transfec- tion code Expected Calculatedmental found found found molecules produced tion of and ratio speciesmass - 2LYS mass (%) (%) (%) (%) 1 Only A AA 146521 146503 100 vector I1 Only G BB 144032 144087 100 vector II 1 Only H CC 144647 144656 100vector III 2 Vector I M AA 146521 146518 51 45 4 and II (I:II = 1:1) BB144032 144030 2 Vector I N AA 146521 146509 88 9 3 and III (I:III = 1:1)CC 144647 144633 U AA 146521 146522 47 48 5 (I:III = 1:5) CC 144647144643 2 Vector II nd BB and III CC 3 Vector I, II O AA 146521 146525 664 30 and III (I:II:III = BB 144032 144032 1:1:1) CC 144647 144650 V AA146521 146531 8 81 9 2 (I:II:III = BB 144032 144043 1:1:10) CC 144647144654 nd = not done.

It was observed that transfections A, G and H resulted in formation ofhomodimers only, and 100% of bivalent monospecific AA, BB or CC wasretrieved from cells transfected with any one of vectors I, II or III(FIG. 4). Although this was to be expected and previously demonstratedfor transfection A, it is actually now shown for the first time thathomodimerisation of CH3-engineered Ig heavy chains containing either thetriple amino acid substitution of construct 4 (i.e., K392D; D399K,K409D) or the quadruple amino acid substitution of construct 5 (i.e.,E356K, E357K, K439D, K370D) is reported (transfections G and H).

Next, co-expression experiments of two vectors in a single cell wereperformed. Interestingly, transfections M and N show that wildtype andCH3 engineered Ig heavy chains can be co-expressed in a single celltogether with a common light chain resulting in mixtures of two speciesof monospecific antibodies without the presence of undesired bispecificantibodies and with as little as 4-5% contaminating ‘other molecules’present in the mixture. ‘Other molecules’ is defined as all moleculesthat do not have the mass of an intact IgG, and includes half moleculesconsisting of a single heavy and light chain pair. Importantly, thefraction ‘other’ does not include bispecific product. In transfection M,the ratio of AA:BB was close to 1:1 upon transfection of equal ratios ofvector DNA. However, transfection N resulted in an almost 10:1 ratio ofAA:CC. Therefore, this transfection was repeated with adjusted ratios ofDNA (transfection U). Indeed, a 1:5 ratio of vector DNA I:III equalizedthe ratio of AA:CC antibody product in the mixture towards an almost 1:1ratio. Thus, transfections M and U show that it is possible to expresstwo different, essentially pure, monospecific antibodies in a singlecell, without undesired by products (i.e., no abundant presence of AC orhalf molecules A or C) (FIG. 5). The novel CH3 modifications ofconstructs 4 and 5 differ substantially from wildtype CH3 such thatheterodimerization between wildtype and 4, or wildtype and 5, does notoccur, which is advantageous for application in large scale productionof mixtures of monospecific antibodies from single cells.

Analogous to these results, also transfection of two different CH3engineered Ig heavy chains (constructs 4 and 5) are expected to resultin mixtures of two different monospecific antibodies only, withoutfurther undesired species present. It is reasoned that the CH3modifications of construct 4 differ substantially from the CH3modifications of constructs 5 such that heterodimerization does notoccur. In that case, co-expression of CH3-engineered heavy chains ofconstructs 4 and 5, together with wildtype CH3 heavy chains in a singlecell would results in 3 monospecific antibodies only.

Indeed, this was observed to be the case as it was found that also amixture of three pure monospecific antibodies could be obtained byexpression of three different Ig heavy chains, designed to formhomodimers over heterodimers, together with a common light chain in asingle cell, with no contaminations present in the mixture (transfectionO) (FIG. 6). As is clear from Table 3, with equal ratios of vector DNAused during transfection O, no 1:1:1 ratio of AA:BB:CC antibodies wasobtained. Transfections with altered vector DNA ratios (1:1:10,transfection V) demonstrated that ratios of AA:BB:CC in the mixtures canbe steered towards desired ratios. Taken together, these experimentsshow that two or three essentially pure monospecific antibodies can beexpressed in a single cell without undesired by products, offeringadvantages for large scale production of mixtures of therapeuticmonospecific antibodies.

Example 11 Mixtures of 2 Bispecific Antibodies from a Single Cell

Whereas use of CH3-engineered heavy chains for production of singlebispecific antibodies has been reported elsewhere, this experiment wasdesigned to investigate whether it is feasible to produce mixtures of 2different bispecific antibodies from a single cell.

Antibody VH regions with known specificities and known ability to pairwith the human IGKV1-39 light chain (FIG. 3) were used for recloninginto vectors containing constructs 1-3 or 6-7 of Table 1 resulting invectors IV-X (Table 4). Vectors IV-X, each containing nucleic acidsequences encoding the common human light chain as well as an Ig heavychain with different CH3 region and different VH specificity, weresubsequently transfected into cells, either alone to demonstrate thatformation of intact monospecific antibodies was hampered, or incombination with another construct vector to obtain bispecificantibodies or mixtures of two bispecific antibodies. Table 5 depicts thetransfection schedule and results.

TABLE 4 VH specificity inserted in different constructs Antigen VH massCloned in construct Vector VH gene specificity (Da) # IV IGHV 3.23Thyroglobulin 12472 1 (B) V IGHV 3.30 Fibrinogen (C) 12794 2 VI IGHV1.08 Tetanus (A) 13703 2 VII IGHV 3.30 Fibrinogen (C) 12794 3 VIII IGHV1.08 Tetanus (A) 13703 3 IX IGHV 1.08 Tetanus (A) 13703 6 X IGHV 3.23Thyroglobulin 12472 7 (B)

TABLE 5 # different Transfec- Experi- Half Full IgG Bispecific Otherbispecifics Transfec- tion code Expected Calculated mental moleculesfound found molecules produced tion of and ratio species mass - 2LYSmass found (%) (%) (%) (%) 0 vector IV B Half B 144082 144066 40 60 0vector V C Half C 144651 144622 77 23 0 vector VI D Half A 146469 14645923 77 0 vector VII E Half C 144625 144643 76 24 0 vector VIII F Half A146443 146468 64 36 0 vector IX P Half A 146691 146677 82 18 0 vector XQ Half B 143818 143844 58 42 1 Vector IV I (1:1) BC 144367 144352 96 4and V 1 Vector IV J (1:1) BC 144354 144382 96 4 and VII 2 Vector IV, VK(1:1:1) BC + AB 144367 + 144351 + 38 + 47 15 (A + C) and VI 145276145260 S(2:1:1) BC + AB 144367 + 144371 + 42 + 55 3 (BB) 145276 145277 2Vector IV, VII L (1:1:1) BC + AB 144354 + 144346 + 16 + 60 24 (A + C)and VIII 145263 145255 T (2:1:1) BC + AB 144354 + 144385 + 58 + 39 3(BB) 145263 145292

It was previously demonstrated that CH3-engineered Ig heavy chainsencoded by constructs 1 and 2 are still able to form homodimers whenexpressed alone in single cells (WO2009/089004). However, WO2009/089004further reports that CH3 domains that are engineered to comprise triplecharge pair mutations, such as present in construct 3, are no longercapable of forming homodimers when expressed alone. In the presentstudy, these findings were only partly confirmed. Indeed, the results oftransfections B, C and D demonstrated the presence of full IgGs, inaddition to a high proportion of unpaired half molecules, demonstratingsome homodimerization of CH3 domains encoded by constructs 1 and 2.Transfections E and F also resulted in production of full IgGs inaddition to unpaired half molecules, demonstrating that the triplecharge mutations of construct 3 do not fully impair homodimerisation. Itwas furthermore demonstrated that also the ‘knob’ and ‘hole’ CH3variants of constructs 6 and 7 form homodimers (18% homodimers for‘knob-knob’ and 42% homodimers for ‘hole-hole’).

CH3 variants that fully prevent homodimerisation when expressed aloneare preferred, to prevent or minimize undesired byproducts (homodimers)upon co-expression with a second CH3 variant for heterodimerization.Interestingly, the present experiments demonstrate for the first timethat also mixtures of bispecific antibodies can be expressed in singlecells with virtually no homodimers in the mixture. Transfections K and Lclearly show that the expected bispecific species BC+AB are indeedobtained (38%+47% in transfection K, and 16%+60% in transfection L). Inboth transfections a relatively high percentage of undesired halfmolecules was observed (15% half molecule A+half molecule C intransfection K, and 24% half molecule A+half molecule C in transfectionL). The relatively high percentage of half molecules still present wasattributed to low amounts of matching heavy chains of vector IV due tounbalanced expression of heavy chains in a matched pair. Therefore,transfections were repeated with an adjusted ratio of vector DNA, 2:1:1,in transfections S and T. This resulted in equal amounts of IgG heavychains constituting a matched pair and pure mixtures of bispecific IgGwithout the presence of half IgG molecules and with as little as 3%homodimeric BB present. Ideally, this low proportion of contaminatingmonospecific product should be reduced to essentially zero. It istherefore desired to find additional CH3-mutants that would result inmixtures of bispecific antibodies with minimal contaminatingmonospecific antibodies present.

The present study demonstrates for the first time that essentially puremixtures of two bispecific antibodies recognizing 3 different targetepitopes can be produced in a single cell, with minimal presence ofmonospecific antibodies in the mixture.

Example 12 Varieties of Mixtures

As it was demonstrated that production of mixtures of 2 bispecificantibodies recognizing 3 epitopes from a single cell, or production ofmixtures of 2 or 3 monospecific antibodies from a single cell istechnically feasible, we next explored the feasibility of controlledproduction of a variety of other mixtures. A fourth antibody VH regionwith known specificity and known ability to pair with the human IGKV1-39light chain will be used for recloning into vectors containingconstructs 1-3 or 7 of Table 1, resulting in vectors I′, II′, III′ or X′(the ′ indicating a different specificity as compared to correspondingvector numbers). The resulting vectors I′-III′, X′ and IV-IX, eachcontaining nucleic acid sequences encoding for the common human lightchain as well as an Ig heavy chain with different CH3 region anddifferent VH specificity, will subsequently be transfected into cells,in combination with other construct vectors to obtain a variety ofmixtures of bispecific and/or monospecific antibodies. The variety ofmixtures that will be obtained include mixtures of 2 bispecificantibodies recognizing 4 epitopes, 2 bispecific antibodies and oremonospecific antibody, or mixtures of 1 bispecific and one monospecificantibody from a single cell. Table 6 depicts the transfection scheduleand expected results.

TABLE 6 Transfection Expected % Variety of Transfection code Expectedmonospecific Expected % mixture of and ratio species IgG Bispecific 2BsAbs, 4 IV + V + IX + ZA (1:1:1:1) BC + AD 0 50 + 50 epitopes X′ 2BsAbs, 4 IV + VII + IX + ZB (1:1:1:1) BC + AD 0 50 + 50 epitopes X′ 2bsAbs + 1 IV + V + VI + ZC (2:1:1:2) BC + AB + 33 33 + 33 mAb wt′ DD 2bsAbs + 1 IV + V + VI + ZD (2:1:1:2) BC + AB + 33 33 + 33 mAb II′ DD 2bsAbs + 1 IV + V + VI + ZE (2:1:1:2) BC + AB + 33 33 + 33 mAb III′ DD 1bsAb + 1 IV + V + wt′ ZF (1:1:2) BC + DD 50 50 mAb 1 bsAb + 1 IV + V +II′ ZG(1:1:2) BC + DD 50 50 mAb 1 bsAb + 1 IV + V + III′ ZH(1:1:2) BC +DD 50 50 mAb 1 bsAb + 1 IV + VII + wt′ ZI (1:1:2) BC + DD 50 50 mAb 1bsAb + 1 IV + VII + II′ ZJ (1:1:2) BC + DD 50 50 mAb 1 bsAb + 1 IV +VII + III′ ZK (1:1:2) BC + DD 50 50 mAb 1 bsAb + 1 IX + X + wt′ ZL(1:1:2) AB + DD 50 50 mAb 1 bsAb + 1 IX + X + II′ ZM (1:1:2) AB + DD 5050 mAb 1 bsAb + 1 IX + X + III′ ZN (1:1:2) AB + DD 50 50 mAb

Although, theoretically, production of all mixtures should be feasible,it is known from previous work by others that large scale production ofclassical knob-into-hole variants is hampered by instability issues.Mixtures resulting from transfections ZA, ZB, ZL, ZM and ZN are thusexpected to become problematic when transferred to larger scaleproduction.

Thus, the current set of constructs present in Table 1 would not allowproduction of all theoretical mixtures from single cells at a largerscale, as knob-into-hole variants are reported to be unstable, and itcannot be excluded that CH3 domains comprising a ‘knob’ or a ‘hole’ willdimerize with either charge variants or wildtype CH3 domains. It is thusdesired to design new CH3-variants that are engineered to preferentiallyform homodimers heterodimers only and which will not homo- orheterodimerize with constructs 1-5 of Table 1 as to allow forco-expression in single cells.

Example 13 Identification of Novel Charge Pair Mutants

The objective of this study was to engineer the IgG CH3 region to resultin the production of only heterodimers or only homodimers upon mixedexpression of different IgG heavy chains in a single cell, wherein thenovel engineered CH3 domains will not homo- or heterodimerize with knownengineered CH3 domains, or with wildtype CH3 domains. Therefore, as afirst step in identifying novel engineered CH3 domains that would meetthe criteria, many interface contact residues in the IgG CH3 domain werescanned one by one or in groups for substitutions that would result inrepulsion of identical heavy chains i.e., reduced homodimerformation—via electrostatic interactions. The objective was to obtain alist of residues that, when substituted by a charged residue, wouldresult in repulsion of identical chains such that these mutations may beused to drive homo- and/or heterodimer formation upon mixed expressionof different IgG heavy chains, whereby the obtained full length IgGs arestable and are produced with high proportions. In a follow up, theidentified substitutions will be used to generate bispecific antibodiesor mixtures of bispecific or monospecific, antibodies by engineeringmatched pairs of CH3 residues in one or more IgG heavy chains—CH3regions. Additionally, newly identified charge mutant pairs may becombined with existing pairs, such that multiple nucleic acid moleculesencoding different heavy chains, all carrying different andcomplementing CH3 mutations, can be used for expression in cells suchthat mixtures of monospecific antibodies only, or bispecific antibodiesonly, or mixtures of defined monospecific and bispecific antibodies canpreferentially be obtained. The residues to be tested in the presentstudy are contact residues as previously identified (Deisenhofer J.,1981; Miller S., 1990; Padlan, 1996, Gunasekaran, 2010). The rationalefor this approach is that repulsive charges are engineered into eachavailable pair of contacting residues. Samples are subsequently analyzedon non-reducing SUS-PAGE to identify pairs in which dimer formation isreduced, as visualized by the presence of bands of approximately 72 kD.All available pairs will be screened as single mutations or incombination with a single other mutation as the repulsive electrostaticinteraction between one non-matching pair may or may not be sufficientto result in sufficient amounts of half-molecules for detection by thismethod, the mutations are also combined.

Amino acid substitutions were introduced in construct vector MV1057 byGeneart according to the table 7 and expression of constructs wasperformed by transfection in HEK293T cells, according to standardprocedures. IgG expression levels were measured in Octet. Whenproduction failed twice, the mutation was considered to be detrimentalto expression and the mutation was not pursued further.

TABLE 7 list of amino acid substitutions in the various constructs thatwere made (EU numbering) Effect on homodimer formation (− = no effect;+++ = max. AA substitutions in inhibition; NT = not CH3 construct #tested on gel) Q347K 8 − Y349D 9 +− Y349K 10 +− T350K 11 − T350K, S354K12 +− L351K, S354K 13 +− L351K, T366K 14 ++ L351K, P352K 15 +− L351K,P353K 16 ++ S354K, Y349K 17 ++ D356K 18 − E357K 19 − S364K 20 ++ T366K,L351K 21 ++ T366K, Y407K 22 +++ L368K 23 NT L368K, S364K 24 ++ N390K,S400K 25 +− T394K, V397K 26 + T394K, F405K 27 +++ T394K, Y407K 28 +++P395K, V397K 29 +− S400K 30 − F405K 31 +++ Y407K 32 ++ Q347K, V397K,33 + T394K Y349D, P395K, 34 + V397K T350K, T394K, 35 NT V397K L351K,S354K, S400K 36 + S354K, Y349K, 37 +− Y407K T350K, N390K, 38 +− S400KL368K, F405K 39 ++ D356K, T366K, 40 +++ L351K Q347K, S364K 41 +++ L368D,Y407F 42 + T366K 43 + L351K, S354K, 44 + T366K Y349D, Y407D 45 + Y349D,S364K, 46 + Y407D Y349D, S364K, 47 + S400K, T407D D399K 48 +− D399R 49+− D399H 50 +− K392D 51 +− K392E 52 +− K409D 53 +

Supernatants containing ≧5 μ/ml IgG were analyzed in SDS-PAGE and IgGwas purified using protein A. The proteins were stained using colloidalblue. Homodimers were visible as a band of approximately 150 kD. Smallerbands of approx 75 kD represented the presence of half molecules (seenegative control: K392D, 1(409D). Blots are shown in FIG. 7.

The results of SDS-PAGE gels were analyzed and scored as presented intable 7, right hand column. A number of residues were consideredpromising for further testing in combination, including residues Q347,S354, Y349. L351, K360, T366, T394, and V397. The choice was based onhigh scores in the inhibition of formation of homodimers combined withthe availability of contacting residues that can be modified withoutrunning into issues such as other non-complementary charges. Forexample, it is known that residues F405 and Y407 have multipleinteractions at the CH3-CH3 interface, including interactions withresidues that are already charged, which may be problematic afterintroduction of multiple charge mutations among these interactingresidues (see Table A). New constructs were made in vector MV1057 (Table8), and antibody VII regions with known specificities and known abilityto pair with the human IGKV1-39 light chain were used for recloning intovectors containing these new constructs (see Table 9) such thatcombinations could further be tested. Table 10 depicts the transfectionschedules and results.

TABLE 8 AA substitutions in CH3 construct # L351K 61 T394K 62 L351D 63T366D 64 S354D, Y349D 65 V397D 66 K360D 67

TABLE 9 VH specificity inserted in different constructs Antigen VH massCloned in construct Vector VH gene specificity (Da) # XI IGHV 1.08Tetanus (A) 13703 8 XII IGHV 1.08 Tetanus (A) 13703 17 XIII IGHV 1.08Tetanus (A) 13703 43 XIV IGHV 1.08 Tetanus (A) 13703 61 XV IGHV 1.08Tetanus (A) 13703 62 XVI IGHV 3.30 Fibrinogen (C) 12794 63 XVII IGHV3.30 Fibrinogen (C) 12794 64 XVIII IGHV 3.30 Fibrinogen (C) 12794 65 XIXIGHV 3.30 Fibrinogen (C) 12794 66 XX IGHV 3.30 Fibrinogen (C) 12794 67

TABLE 10 Transfec- AA AC CC Half A Half C Transfec- tion code Expectedfound found found found found other tion of (ratio) species (%) (%) (%)(%) (%) (%) XIII + XVI ZO (1:1) AC 0 69 7 24 0 0 ZT (3:1) AC 10 45 16 270 0 ZU (1:1) AC 5 61 10 13 0 0 ZV (1:3) AC 3 61 23 13 0 0 ZW (1:1) AC 088.3 2.4 7 0 2.3 XIV + XVII ZP AC 30 52 13 0 0 5 XII + XVIII ZQ AC 4 5133 2 1 8 XV + XIX ZR AC 20 42 11 0 1 26 XI + XX ZS AC 34 41 15 0 0 10

Combinations of CH3 variants were expressed, and analyzed in SDS-PAGE(data not shown) and in native mass spectrometry (MS). Results aresummarized in Table 10. The ZO transfection resulted in the highestproportion of heterodimers in the mixtures (69% AC). Interestingly, inthe ZO transfection, the AA homodimer was not present whereas the CChomodimer comprised a small proportion (7%). Mass spectrometric analysisunveiled that the remaining protein in the mixture consisted of half Amolecules, probably resulting from unequal expression of the A and Cheavy chains. The raw MS data from transfection sample ZO are shown inFIG. 8. Surprisingly, whereas transfection ZO resulted in fair amountsof bispecific product, the reverse charge pair of transfection ZP(L351K/T366′D versus T366K/L351′D of ZO) did not result in similarresults, and only 52% of bispecific product was observed, withconsiderable amounts of the two homodimers being present (30% AA and 13%CC). An explanation for this may be that the negatively charged Dstructurally closely resembles T, hence the T366D may not be potentenough to repulse itself and T366D will thus still form homodimers, aswas indeed observed.

It can be envisaged that subtle variants of the newly found T366K/L351′Dpair (e.g. by testing all permutations including new constructs T366E,and L351E) may result in similar percentages of BsAbs.

Example 14 HADDOCK for Design of New CH3 Mutants to Drive EfficientHeterodimerization

As described in example 13, the newly found charge pair T366K/L351′Dincreases the proportion of heterodimers in the mixture (69%) with asmall fraction of undesired CC homodimers (7%) (L351D/L351′D) and asubstantial fraction of half A molecules (24%) ‘contaminating’ themixture. In this example, an in silico approach was used to generatefurther insight in amino acid residues involved CH3 interfaceinteractions, to test complementary substitutions in opposing CH3regions and to find novel CH3 pairs containing complementarysubstitutions that further increase efficient heterodimerization whilepreventing efficient formation of homodimers of the two heavy chains.

HADDOCK (High Ambiguity Driven protein-protein DOCKing) is aninformation-driven flexible docking approach for the modeling ofbiomolecular complexes. HADDOCK distinguishes itself from ab-initiodocking methods in the fact that it encodes information from identifiedor predicted protein interfaces in ambiguous interaction restraints(AIRs) to drive the docking process, (de Vries it al., 2010). The inputfor the HADDOCK web server consists of a protein structure file, whichcan be a crystal structure, NMR structure cluster or a modeledstructure. After the docking or refinement. HADDOCK returns a so-calledHADDOCK score, which is a weighted average of VanderWaals energy,electrostatic energy, buried surface area and desolvation energy. TheHADDOCK score can be interpreted as an indication of binding energy oraffinity, even though a direct translation to experimental data is oftenhard to achieve. In addition to this, HADDOCK provides structure filesfor the ‘top four’ structures that resulted from the docking run. Thesestructure files can be downloaded and visualized, enabling the detailedanalysis of the interactions of the individual residues.

In this example, the interactions between the CH3-domains of the IgG1heavy chains were studied. A high-resolution crystal structure of the Fcpart of the IgG (structure 1L6X) was used as starting structure(http://www.rcsb.org/pdb/explore/explore.do?structureId=116x; Idusogie,E. E. et al., J. I., 2000(164)4178-4184).

In example 13, it was found that co-transfection of vectors XIII and XVIresulted in the formation of the CC homodimeric contaminant (Table 10),HADDOCK was used to search for additional mutations to the T366K/L351′Dpair that prevent homodimerization.

The HADDOCK output consists of a set of calculated energies, a HADDOCKscore (which is a weighted average of the energies) and four structurefiles corresponding to the four lowest-energy structures found by theprogram. The HADDOCK-scores are used to compare different structures;the other energies are merely used to get an indication about what ishappening in the structures (e.g. good electrostatic interactions,smaller buried surface, high Van der Waals energy). The lower theHADDOCK score, the better. For, each mutation pair, the scores werecalculated for the AA, AB and BB dimers.

Sets of mutation pairs from example 12 were run in HADDOCK to seewhether the calculated energies would correlate to the experimentaldata. Table 11 presents all theoretical energies, which are visualizedin FIG. 9.

TABLE 11 Buried HADDOCK VdW Electrostatic Desolvation surface Constructcombinations Score energy energy energy area Wildtype-wildtype −208.2−62.8 −773 9.2 2505.8 1-2 (E356KD399K-K392DK409D) −225.8 −56.4 −862 32458.3 2-2 (K392DK409D-K392DK409D) −180.3 −67.9 −562.1 0.1 2312.5 1-1(E356KD399K-E356KD399K) −176.7 −75.5 −469.3 −7.3 2349.6 1-3 (E356KD399K-−220.6 −67.9 −793.8 6.1 2499.8 K392DK409DK439D) 3-3 (K392DK409DK439D-−150.1 −76.6 −387.6 4.1 2261.2 K392DK409DK439D) 6-7(T366W-T366SL368AY407V) −221.3 −65.8 −735.5 −8.3 2509.0 6-6(T366W-T366W) 1916.9* 2072.3 −681.3 −19.2 2499.9 7-7 (T366SL368AY407V-−191.9 −55.0 −683.2 −0.2 2427.2 T366SL368AY407V) 43-63 (T366K-L351D)−210.6 −64 −758.4 5.1 2456.5 43-43 (T366K-T366K) −191.7 −71.2 −634.1 6.32533.5 63-63 (L351D-L351D) −212.5 −60.4 −774 2.6 2445.6 *this value isunusually high due to high VanderWaals energy score, probably due tosteric clash of T366W/T366′W

With 2 wildtype C83 domains, the HADDOCK scores are the same for AA, ABand BB because the A and B CH3 regions are identical. In most othercases, the AB pair has the lowest score, which is as expected. For theT366K/L351D pair the BB score is slightly better than the AB score(−210.6 vs. −212.5), hut this difference is within the error of thecalculations. Using HADDOCK, the structures of the heterodimers of thesepairs were visualized. For example, the construct combinations 1-2, 1-1and 2-2 are presented in FIG. 10. From these visualizations it isapparent that salt bridges are formed in the heterodimer (FIG. 10A lefthand panel) whereas electrostatic repulsion occurs between residues ofidentical chains (FIGS. 10B and C, middle and right hand panel). Thehigher HADDOCK scores for the homodimers can thus be explained by theelectrostatic repulsion of the mutated interface residues. Theseresidues have to bend away from each other and don't have interactionwith residues on the other chain, causing a drop in the affinity.

Table 11 and FIG. 9 confirm what was observed in example 13. TheT366K/L351′D AC heterodimer and the L351D/L351′D CC homodimer form witha similar energy, explaining the presence of both the heterodimer andhomodimer in the mixture. The T366K/T366′K AA homodimer, on the otherhand, is barely detectable in the mixture although T366K half Amolecules are present. Table 11 and FIG. 9 indeed show that the HADDOCKscore for the T366K/F366′K AA homodimer is higher than the score for theAC heterodimer; hence formation of this homodimer is energetically lessfavorable.

Example 15 366/351 Variations

In example 13, it is hypothesized that alternatives for the T366K/L351′Dmutant charge pair can be designed that may have similar results interms of percentage of bispecific antibodies in the mixture.Alternatives may include substitutions T366R, T366D, T366E, L351E, L351Kand L351R. The proportion of CC homodimers of L351D/L351′D may bediminished by creating variants of the 366/351 pair. All possiblemutation pairs were run in HADDOCK and the resulting scores arepresented in Table 12 and visualized in FIG. 11.

TABLE 12 Electro- Buried Construct HADDOCK VdW static Desolvationsurface combinations Score energy energy energy area T366K-L351D −210.6−64 −758.4 5.1 2456.5 T366K-T366K −191.7 −71.2 −634.1 6.3 2533.5L351D-L351D −212.5 −60.4 −774 2.6 2445.6 T366K-L351E −216.9 −55.7 −854.79.8 2532.7 L351E-L351E −217.9 −65.5 −802.2 8 2532 T366R-L351D −210.5−68.8 −760.8 10.4 2514.5 T366R-T366R −201.8 −77.4 −626.4 0.9 2608T366R-L351E −225.8 −56.2 −874.8 5.4 2579.2 T366D-L351R −211.2 −71.3−723.6 4.8 2455.6 T366D-T366D −198.1 −58.1 −713.4 2.1 2477 L351R-L351R−220.7 −75.5 −806.5 16.1 2552.2 T366D-L351K −223.9 −62.1 −810.1 0.32487.8 L351K-L351K −224.4 −75.6 −812.1 13.6 204.5 T366E-L351R −222.3 −69−783 3.4 2557.2 T366E-T366E −201.9 −57.6 −741 4 2487.5 T366E-L351K−215.9 −58.4 −808.9 4.3 2486

When looking at the HADDOCK scores, it was observed that some of themutations have a similar ‘pattern’ when compared to T366K/L351′D. Formost permutations the AA homodimer was found to have a higherHADDOCK-score than the AB heterodimer, but the BB homodimer appeared asfavorable as the AB heterodimer. Even though the 351 residue is known tobe a ‘neighbor’ to itself on the other chain, i.e. residue 351 of chainA pairs with residue 351 of chain B at the CH3-CH3 interface, there isbarely a negative influence of the identical charges when the BB dimeris formed. Looking at the L351D/L351′D structure this is explained bythe aspartic acids bending away from each other and the stabilizinginfluence of at least the naturally occurring Arginine at position 355and also some stabilization of negative charge by the naturallyoccurring Serine at position 354 (see FIG. 12A). Mutation of theseresidues (S354A and R355D) provides only little improvement. From FIG.12B it is clear that the backbone-hydrogen of A354 causes stabilizationof the homodimer. From this series, the T366R/L351′E pair seems to bethe most favorable, with the lowest HADDOCK score for the bispecificmolecule.

Example 16 Mutations Around T366K/L351′D

In the series of HADDOCK analyses in this example, the T366K/L351′D orT366K/L351′E pair were taken as a starting structure. In order toidentify additional mutations that would further increase the predictedpercentage of bispecifics of these A and B chains, additional mutationson the B-chain were used to calculate the HADDOCK-scores and energies.When the structure of the CH3 domain is studied using a viewer forvisualization of protein structures at a molecular level (YASARA,www.yasara.org), one can calculate the distances between individualresidues. While doing so, it was observed that the two residues Y349 andL368 are neighboring residues that may contribute positively ornegatively to dimer interactions and these have been mutated in thisexample in addition to the L351D mutation—to study the result on dimerformation of the home- and heterodimers (see FIG. 13). Both residuesseem to add to the stability of the heterodimer (lower HADDOCK scores)as well as to the destabilization of the BB dimer (higher HADDOCKscores). Glutamic acids (E) on positions 349 and 368 seem to be morefavorable than aspartic acids (D). Thus, introduction of a second aminoacid substitution in the B-chain, comprising already the amino acidsubstitution at position 351, seems to favor heterodimerization further.

In a next set of HADDOCK analyses, the T366K/L351′D pair was again takenas starting structure. In addition to the substitutions in the B chainthat further increased heterodimerization (i.e. Y319DIE and L368E),additional mutations were added to the A-chain which already comprisesthe T366K substitution. As shown in FIG. 14, there are several mutationpairs that seem favorable towards the formation of bispecificheterodimers. In the T366K-L351K/L351′D-Y349′D pair, all four mutatedresidues are involved in the heterodimeric pairing, which is not de casefor T366K-L351K/L351′E-L368′E in which K351 is not directly involved inthe binding. However, the HADDOCK-score for this latter heterodimer−228.9; significantly lower than the −214.2 for the T366K/L351′E-L368′E,which can be explained by hydrogen bonding interactions of the K atposition 351 (see FIG. 15). The T366K-L351K/L351′D-Y349′D pair may befurther improved by the R355′D mutation in the B-chain, which results ina higher BB-HADDOCK score, but also the AB HADDOCK score is slightlyhigher. Overall the additional L351K results in lower AB scores andsimilar AA and BB scores when compared to the sole T366K mutation in theA chain. Theoretically this would result in higher amounts of bispecificheterodimers in the samples.

As is apparent from FIG. 11, having an R rather than a K at position 366may be more potent in driving heterodimerization. Therefore, some of theHADDOCK analyses shown in FIG. 13 were repeated but now with T366Rrather than T366K the A-chain. It was demonstrated that it is notfavourable to combine an 11366 in chain A with double mutations in chainB (FIG. 16). This may be due to the large size of this residue,interfering with other interface interactions, even though all theexpected salt-bridges with R366 are present in the structures. Also, theHADDOCK score for the AA homodimer is lower for R366 than for K366,which also doesn't contribute favorably to heterodimer formation.Therefore no further HADDOCK analyses were performed using R366 in theinterface.

A total of 14 best performing pairs, according to HADDOCK predictions,have been selected (see Table 13 and FIG. 17). In some pairs, an R355Dsubstitution is included to remove the stabilizing, influence of thenaturally occurring R355 on the L351/L351′D interaction.

TABLE 13 HADDOCK HADDOCK HADDOCK Construct combinations Score AB ScoreAA Score BB Wildtype-wildtype −208.2 −208.2 −208.2 T366K-L351D −210.6−191.7 −212.5 T366K-L351E −216.9 −191.7 −217.9 T366R-L351E −225.8 −201.8−217.9 T366E-L351R −222.3 −201.9 −220.3 T366K-L351DY349E −215.9 −191.7−190 T366K-L351DL368E −223.3 −191.7 −198.9 T366K-L351EY349E −214.5−191.7 −187.5 T366KL351K-L351D −233.2 −205 −212.5 T366K- −207.5 −191.7−179.5 L351DY349EL368E T366KL351K- −255.2 −205 −204.3 L351DY349DT366KL351K- −227.2 −205 −190 L351DY349E T366KL351K- −243.9 −205 −198.9L351DL368E T366KL351K- −233.6 −205 −211.9 L351DR355D T366KL351K- −242.8−205 −183.5 L351DY349DR355D T366D-L351KY349K −237.9 −198.1 −228.4

Example 17 In Vitro Expression of Bispecifics Using CH3 Mutants Based onHADDOCK Predictions

The analysis in example 16 suggested that some CH3 variants withadditional mutations around the T366K/L351′D pair would yield mixtureswith higher proportions of the bispecific component and potentiallylower proportions of the homodimeric component. These best performingpairs were selected for production and further analysis. In addition,the constructs T366R and L351E were also generated. Table 14 lists theconstructs that were made and which were used for rearming antibody VHregions with known specificities and known ability to pair with thehuman IGKV1-39 light chain. Expression of the IgGs that contain theindividual constructs was previously reported in example 13, and wasrepeated for the constructs as listed in Table 14. Aim was to assesswhich of the constructs homodimerize in the absence of a matchingheterodimerization partner. Ideally, high percentages of half bodieswould be formed and low percentages of homodimers. As a control,constructs containing previously reported charge mutations andconstructs containing the previously reported knob-in-hole mutationswere also used for expression as whole IgG by recombinant cells. ProteinA purified supernatants were analyzed in SDS-PAGE; results were analyzedand scored as presented in Table 14

TABLE 14 Construct % half AA substitutions in CH3 # % IgG moleculeE356K, D399K 1 64.2 35.8 K392D, K409D 2 30.9 69.1 K392D, K409D, K439D 324.5 75.5 T366W 6 27.6 72.4 T366S, L368A, Y407V 7 58.6 41.4 T366K 4332.9 67.1 L351D 63 89.8 10.2 T366D 64 89.6 10.4 T366K, L351K 68 34.765.3 L351D, L368E 69 83.7 16.3 L351E, Y349E 70 67.8 32.2 L351D, Y349E 7179.7 20.3 L351D, R355D 72 100 — L351D, Y349E, L368E 73 79.3 20.7 L351D,Y349D 74 88.6 11.4 L351D, Y349D, R355D 75 89.9 10.1 L351K, L368K 76 56.643.4 L351R 77 100 — T366E 78 44.4 55.6 T366R 79 29.6 70.4 L351E 80 100 —

The results of co-expression of a common light chain and two differentheavy chains carrying the amino acid substitutions of constructs shownin Table 14 or heavy chains carrying the amino acid substitutions ofprevious constructs are presented in Table 15. Expression of twodifferent heavy chains comprising the amino acid substitutions T366K andL351′D:L368′E respectively resulted in approximately 87% of thebispecific AB heterodimer in the mixture with no AA or BB homodimerspresent (combination nr. 3 of Table 15). About 12% half molecules (halfA) comprising the T366K substitution was observed. Furthermore, it wasfound that the percentage of bispecific AB heterodimer increased whenthe additional amino acid substitution L351K was introduced in the firstheavy chain. For example, coexpression of two different heavy chainscomprising the amino acid substitutions T366K:L351K (and L351D:L368′Erespectively resulted in approximately 92% of bispecific AB heterodimerwhereas and BB homodimers are essentially absent in the mixture(combination nr. 12 of Table 15). Combinations 10 and 11 also resultedin favorable distributions of high percentages heterodimers andvirtually absence of homodimers. The absence of homodimers isadvantageous, because the fraction containing the intact IgG moleculesis composed of AB heterodimer only. For purification and subsequenttherapeutic application, the half molecules can be removed by standardapproaches such as size exclusion chromatography. Hence, applying thesenewly identified charge mutants in the production process for generatingbispecific antibodies provides advantages over known charge mutants andknobs-into-holes mutants where the presence of ‘contaminating’homodimeric antibodies is not excluded. In addition, theT366K/L351′D:L368′E and T366K:L351K/L351′D:L368′E charge pairs have anadditional advantage over the previously describedE356K:D399K/K392′D:K409′D and E356K:D399K1K392′D:K409′D:K439′D chargereversal pairs, in that the previously described charge variants arebased on the reversal of existing charges within the CH3-CH3 interfacewhereas the newly identified charge variants are adding additionalcharge pairs (charge-charge interactions) to the CH3-CH3 interface. Theintroduction of additional charge pairs in the CH3-CH3 interface mayfurther increase the stability of the interface and thereby of theintact antibody. The same holds true for the mutations used incombinations nrs. 4, 5, 6, 9, 10, and 11, which also resulted infavorable proportions of bispecific heterodimer with exceedingly lowproportions of AA and BB homodimers present in the mixtures.

TABLE 15 Combination of 2 different chain A*/mutations chainB**/mutations % AA % AB % BB % half A % half B heavy chains (construct#) (construct #) found found found found found 1 T366E (78) L351R (77) 381 2 13 0 2 T366K (43) L351D (63) 0 88 3 9 0 3 T366K (43) L351D, L368E(69) 0 87 0 12 0 4 T366K (43) L351E, Y349E (70) 2 85 0 11 0 5 T366K (43)L351D, Y349E (71) 2 92 1 5 0 6 T366K (43) L351D, Y349E, L368E (73) 0 961 4 0 7 T366K, L351K (68) L351D (63) 0 77 12 10 1 8 T366K, L351K (68)L351D, R355D (72) 0 79 8 10 1 9 T366K, L351K (68) L351D, Y349D, R355D(75) 1 93 2 4 1 10 T366K, L351K (68) L351D, Y349D (74) 1 95 1 3 0 11T366K, L351K (68) L351D, Y349E (71) 1 95 0 3 1 12 T366K, L351K (68)L351D, L368E (69) 0 92 0 8 0 13 T366K (43) L351E (80) 0 70 10 18 2 14T366R (79) L351E (80) 4 38 36 21 1 15 T366D (64) L351K, L368K (76) 3 922.5 2.5 0 16 T366D (64) L351R (77) 30 69 1 0 0 *chain A carriesspecificity of MF1337 (= tetanus toxoid); **chain B carries specificityof MF1122 (= fibrinogen)Native MS

Native MS was performed on all bispecific samples. The obtained graphswere analyzed to determine the relative ratio's of the present speciesin two ways: by peak height and by peak area. Peak area is the morescientifically correct way of analysis, but since all previous analysesfor other studies were done based on peak height, both methods wereincluded in the analysis, for comparison purposes. The differencesbetween the methods were within the error of measurement, and thereforeonly the peak area values were used for future measurements. Two typicalspectra are shown in FIG. 18. An overview of the results is showngraphically in FIG. 19, the numerical values can be found in Table 15.In about half of the samples the total contamination of monospecific IgGis less than 5%, and only in three cases it is >10% while for wt IgG itis expected to find about 50% of monospecific IgG in the mixture.

A panel of ten combinations of 2 different heavy chains was selectedfrom Table 15 for further analyses. These ten combinations includedcombinations 1, 2, 3, 4, 5, 6, 9, 10, 11 and 12 (Table 15). Selection ofthese ten was based on low percentages of homodimers present in themixtures as determined by nMS, but also based on their overallphysico-chemical properties, including production yields, SDS-PAGE, aswell as the number of mutations present in the CH3 domain.

Example 18 IgG Stability Analyses

In this study, a series of CH3 mutation pairs that resulted in highproportions of bispecific heterodimers in the intact IgG fraction andvery low amounts (<5%) of parental IgGs will be further analyzed forstability of the Fc part of the IgG molecule. The mutated CH3 domainsthat are used to promote the heterodimerization of the heavy chains mayhave unexpected destabilizing effects on the FC region of the IgG, thatmay result in undesirable properties such as a reduction of in vivo halflife, reduction in effector function and/or an increase inimmunogenicity. The newly identified charge pairs will be compared towildtype bispecifics and a bispecific containing previously identifiedcharge mutations (chain A comprising construct 1 and chain B comprisingconstruct 2). All bispecifics in this study will contain the same heavyand light chain variable regions, ensuring that the observed effects arecaused by mutations in the Fc-part of the molecule and not by variationin the variable regions.

A series of stability studies will be performed on these bispecifics.These studies include spectroscopic (UV-Vis absorbance, fluorescence andlight-scatter) and microscopic (light and fluorescence microscopy withNile Red staining) analyses that provide information on the aggregationstate, of the CH3 variants.

The UV-Vis absorbance spectra will be recorded with a double beam, twomonochromators Cary 300 Bio spectrophotometer at 25° C. The spectra willbe monitored between 250 and 400 nm using a path length of 1 cm. Theabsorbance at wavelengths of 320 nm and longer provides information onthe aggregation state of the IgG.

Intrinsic fluorescence spectra will be monitored at 25° C. using aFluoroMax spectrofluorimeter. The fluorescence method will be optimized.The fluorescence emission will provide information on conformation andaggregation properties. 90° light-scattering spectra will be monitoredat 25° C. using a FluoroMax spectrofluorimeter by running a synchronousscan (λ_(em)=λ_(ex)) between 400 nm and 750 nm with an integration timeof 0.01 s. Excitation and emission slits will be optimized. For example,right angle light-scattering can distinguish between IgG samples thathave no and 5% dimers.

For fluorescence microscopy with Nile Red staining, just prior tomeasurements, Nile Red in ethanol will be added to the sample. Thesamples will be filled in a microscopy slide and analyzed byfluorescence microscopy. Particles will be counted. The lower size limitof the particles that can be observed by fluorescence microscopy isapproximately 0.5 μm.

Application of stress such as temperature, pH, mechanical stress ordenaturants on proteins might result in a conformation change (e.g.unfolding) and/or aggregation. As it was previously reported thatcharge-engineered bispecific antibodies have reduced melting temperatureof the modified CH3 (Gunasekaran 2010), these studies aim todiscriminate between the novel charge mutants of the present inventionand existing known charge mutants.

Thermo-stability studies using the Octet are explored, both with ProteinA biosensors and by using FcRn binding to IgG. To examine the thermalstability of CH3-engineered IgGs, the samples will be incubated at aconcentration of 100 ug/ml (in PBS) at 4, 50, 55, 60, 65, 70 and 75° C.for 1 hour using a PCR machine. Following this the samples will becooled down slowly during a period of 1.5 minutes to 25° C. and kept atthis temperature for 2 hours, after which they will be stored overnightat 4° C. Precipitated antibodies will be removed by centrifugation,after which the total IgG concentration of soluble antibodies will bedetermined by Octet using the protein A Biosensor (1/10 dilution inPBS). Assays that measure binding of the CH3 engineered IgG to theOctet, are being explored. Either protein L biosensors are used to bindthe light chain of IgG to the sensor, followed by incubation with FcRnin solution, or anti-penta-HIS biosensors are used to bind His-taggedFcRn protein, followed by incubation with the IgG of interest. Thesemethods may be more sensitive than using the protein. A Biosensor andcan also be used for thermal stability studies. All samples will also beanalyzed for serum stability. Briefly, (engineered) IgG samples will beincubated at 37° C. in human serum, control samples will be kept at 4°C. After 1, 2, 3 and 4 weeks, samples are centrifuged to removeprecipitated IgG. Subsequently the sample is titrated inantigen-specific ELISA to determine the relative amounts of functionalIgG. Purified control antibody freshly spiked in human serum will beused as a reference.

Example 19 Stability Analyses

In previous experiments, high percentages of bispecific antibodies wereObtained by co-expression of two different heavy chains comprising CH3mutations, and a common light chain (example 1.7).

A panel of eight combinations of 2 different, heavy chains was selectedfrom Table 15 for further analyses. These eight combinations includedcombinations 3, 4, 5, 6, 9, 10, 11 and 12 (Table 15). In this study,these eight combinations were analyzed, with a strong focus on stabilityof the Fc part of the IgG. As controls, wildtype bispecifics without CH3mutations) and/or bispecifics based on previously reported. CH3 chargemutations were included. Note that for wildtype bispecifics, 2 heavychains and the common light chain are co-expressed without means forpreferential steering towards heterodimers. These ‘wildtype bispecifics’thus represent a mixture of AA, AB and BB. All bispecifics in this studywere designed to carry the same VH/VL-combinations, ensuring that theobserved effects are caused by mutations in the Fc-part of the moleculeand not by variation(s) in the Fab parts.

It was hypothesized that the mutational pairs that were used to promotethe heterodimeric pairing of the two different heavy chains could beassociated with unexpected structural or otherwise destabilizing effectson the Fc region of the IgG. This could subsequently result in undesiredissues that would hamper further clinical development, such as areduction of in vivo half life, a reduced effector function and/orincreased immunogenicity due to the presence of these mutations.

Thermo Stability

Application of stress such as increases or decreases in temperaturemight result in a conformation change (e.g. unfolding) and/oraggregation of proteins. To examine the thermal stability ofCH3-engineered IgGs, the bispecific molecules from combinations 3-6 and9-12 (Table 15), as well as wildtype bispecifics and bispecificmolecules obtained when using constructs 1 and 2(E356K:D399K/K392D′:K409D′ combination, also dubbed ‘charge reversal.’pair) were incubated at a concentration of 100 μg/ml (in PBS) at 4, 60,62.5, 65, 67.5, 70 and 72.5° C. for 1 hour using a PCR machine.Following this the samples were cooled down slowly during a period of 15minutes to 25° C. and kept at this temperature for 2 hours, after whichthey were stored overnight at 4° C. The next day, precipitatedantibodies were removed by centrifugation (18,000 rpm; 4° C., 20 min),after which the total IgG concentration of soluble antibodies wasdetermined by Octet using the protein A Biosensor (1/10 dilution inPBS). Results are shown in FIG. 20. It was observed that the control CH3engineered bispecific antibody (the charge reversalE356K:D399K/K392D′:K409D′ combination (triangles)) has a reduced thermalstability as compared to the wildtype bispecific (squares). Thebispecific molecules from combinations 3-6 and 9-12 (diamonds) alsodemonstrated a reduced thermal stability as compared to wildtype.Remarkably, three combinations, however, demonstrated an improvedstability as compared to the control CH3 engineered bispecific antibody.Bispecifics of combinations 9, 10 and 11 are significantly more stablethan the other CH3 engineered (charge reversal) bispecifics and are asstable as wildtype bispecifics at the highest temperature measured.

Freeze-Thaw Stability

To examine the stability of CH3-engineered IgGs upon repetitive freezingand thawing, the bispecific molecules from combinations 3-6 and 9-12(Table 15), as well as wildtype bispecifics and bispecific moleculesobtained when using constructs 1 and 2 (E356K:D399K/K392D′:K409D′combination (charge reversal pair)) were exposed to ten subsequentfreeze-thaw cycles by putting the samples at −80° C. for at least 15minutes until they were completely frozen. Thereafter, samples werethawed at room temperature. When they were completely thawed, thefreeze-thaw cycle was repeated. After 10 freeze-thaw cycles,precipitated antibodies were removed by centrifugation (18,000 rpm; 4T,20 min), after which the total IgG concentration of soluble antibodieswas determined by Octet using the protein A Biosensor (1/10 dilution inPBS). The freeze-thaw stability test was repeated three times. Resultsare shown in FIG. 21. It was observed that the control charge reversalCH3 engineered bispecific antibody seemed to have a slightly reducedstability as compared to the wildtype bispecific. In contrast, thebispecific molecules from combinations 3, 4 and 9 seemed to have aslightly improved stability as compared to the wildtype bispecific.Overall, it can be concluded that the stringent conditions offreeze-thaw cycles do not cause major stability issues for the CH3engineered variants.

In Vitro Serum Stability

To examine the stability of CH3-engineered IgGs in serum kept at 37° C.,the bispecific molecules from combinations 3-6 and 9-12 (Table 15), aswell as wildtype bispecifics and the charge reversal bispecificmolecules were incubated at 37° C. in 10% human serum. Control sampleswere kept in human serum at 4° C. After 1, 2 or 5 days, precipitatedantibodies were removed by centrifugation. Thereafter, the samples weretitrated in a fibrinogen-specific ELISA, to determine the relativeamounts of functional IgG. Purified control antibody freshly spiked inhuman serum was used as reference.

Data of the fibrinogen ELISA show that all samples were quite stable in10% human serum at 37° C. for 5 days. At the lower IgG concentrationbispecific molecules from combinations 4 and 5 seem to be slightly lessstable, especially at T=1 and T=2, but the difference is only minimal atthe end-point of this experiment (see FIG. 22).

Example 20 Further Stability Tests

A further series of analytical methods was used to assess the stabilityof the variant IgGs. Bispecific molecules from combinations 3-6 and 9-12(Table 15), as well as wildtype bispecifics (AA, AB, BB), the individualparental antibodies (AA and BB) and bispecific molecules obtained whenusing constructs 1 and 2 (E356K:D399K/K392D′:K409D′ combination (chargereversal pair)) were used as samples in these stability assays. All IgGswere diluted to 0.2 mg/ml and several stress conditions (2 days at 50°C., 2 weeks at 40° C., 5× freeze-thawing) were applied, aiming to beable to discriminate between the different samples. Of note, these highstress levels resulted in conditions in which one of the parentalantibodies (the BB parental, carrying two 1122 Fabs) as used in allbispecifics became unstable. At 2 days at 50° C., aggregation of thisprotein was detected by UV absorbance. This suggested that this stresscondition may not differentiate between instability of the Fab and theCH3 in the bispecific and data resulting from the 50° C. incubationshould be used cautiously.

The results are summarized in Table 16. Analytical methods that wereused included:

-   -   Fluorescence microscopy with Nile Red (‘Nile Red particles’ in        Table 16); to observe the amount of particles>0.5 μm after        addition of Nile Red dye.    -   UV spectrometry at 350 nm (‘UV 350 nm’); a change in absorption        at wavelengths>320 nm gives information about the aggregation        state of the protein.    -   90° Light scatter at 400 nm (‘LS 400 nm’); a sensitive technique        to observe changes in protein aggregation, e.g. the difference        between monomers and dimers of IgG.    -   Intrinsic fluorescence; the fluorescence wavelength maximum and        intensity of the aromatic residues in a protein change upon        changes in the environment (e.g. unfolding)    -   1,8-ANS fluorescence spectroscopy; 1,8-ANS binds through        electrostatic interactions to cationic groups through ion pair        formation and changes in protein structure and/or conformation        can be detected        UV-VIS Spectroscopy

UV-Vis absorbance spectra were measured at 25° C. with a double beam,two monochromators Cary 300 Bio spectrophotometer from Varian indifferent quartz cuvettes (such as black low volume Hellma cuvettes witha pathlength of 1.0 cm and clear Hellma cuvettes of 0.2 cm×1.0 cm). Thespectra were monitored between 220 and 450 nm using a pathlength of 1.0cm. The absorbance around 280 nm provides information on the proteinconcentration. The region between 320 nm and 450 nm can provideinformation on the aggregation state of the samples.

90° Light-Scattering

The 90° light-scattering spectral method was developed to study proteinaggregation and was performed as described in Capelle, 2005; Demeule,2007a. 90° light-scattering spectra were monitored at 25° C. using aFluoroMax spectrofluorimeter (Spex, Instruments S.A., Inc. U.K.) byrunning a synchronous scan (λ_(em)=λ_(ex)) between 400 nm and 750 nmwith an integration time of 0.01 s. Different slits settings were testedin order to find the optimal conditions. After optimization, the sameslit settings were used for all measurements.

Steady-State Fluorescence Emission

The fluorescence emission of tryptophan, tyrosine and phenylalanineresidues gives information on the local environment of thesefluorophores. Changes or differences in hydrophobicity and/or rigidityare measured. Typically, a more hydrophobic and rigid environment leadsto an increase in the fluorescence intensity and a blue shift of theemission maximum. Intrinsic fluorescence spectroscopy can provideinformation on the current state of the protein and monitor changes inthe physical and chemical properties. More information on thefluorescence of tyrosine and tryptophan can be found in the book ofLakowicz [Lakowicz, 2006].

The fluorescence emission and excitation spectra were recorded at 25° C.in different quartz cuvettes. The samples were excited at differentwavelengths, integration times and slit settings were optimized. Afteroptimization, the same integration times and slit settings were appliedfor all samples.

Fluorescence Microscopy with Nile Red Staining

The Nile Red staining method was developed to visualize proteinaggregates and was performed as described in Demeule et al., 2007b.

The microscopy observations were performed on a Leica DM RXE microscope(Leica Microsystems GmbH, Wetzlar, Germany) equipped with a mercurylamp. The images were acquired with a Sony NEX-5 camera and itsfirmware. The objectives were 10×, 20× and 40×. For microscopyinvestigations slides with a fixed distance of 0.1 mm between the slideand the cover glass were used. The size of the 4×4 grids is 1 min×1 mmand corresponds to 0.1 μl.

1,8-ANS Fluorescence Spectroscopy

1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) is an uncharged smallhydrophobic fluorescent probe (Mw 299.34 Da) used to study both membranesurfaces and proteins.

1,8-ANS is essentially non-fluorescent in water and only becomesappreciably fluorescent, when bound to membranes (quantum yields ˜0.25)or proteins (quantum yields ˜0.7). This property of 1,8-ANS makes it, asensitive indicator of protein folding, conformational changes and otherprocesses that modify the exposure of the probe to water. References on1,8-ANS can be found on the Internet home page of Molecular Probes,www.probes.com.

The fluorescence emission spectra of 1,8-ANS were recorded using aFluoroMax spectrometer. A direct comparison of the 1,8-ANS fluorescencebetween IgGs will not be performed. Each IgG can have different numberof 1,8-ANS binding sites and can therefore not be compared. Inprinciple, the lower the 1,8-ANS fluorescence, the less 1,8-ANSmolecules are bound to the antibody. The changes in the 1,8-ANSfluorescence intensity and emission wavelength due to stress will beevaluated.

TABLE 16 Overview of the different forced degradation results on variousIgG samples after dilution to 0.2 mg/ml.

The colour of the cells indicate the variations between T = 0 and afterstress: dark grey = large change, light grey = small change and nocolour = no change (= stable). *‘combi. #’ refers to the combination ofmutations as listed in Table 15; **very small particles by fluorescencemicroscopy, relevance of these particles unknown; 2d4° C. = 2 days at 4°C.; 2d50° C. = 2 days at 50° C.; 2w4° C. = 2 weeks at 4° C.; 2w40° C. =2 weeks at 40° C.; T0 = start of experiment; 5FT = 5 freeze thaw cycles

Taken together, these data indicate that the various IgG samples areremarkably stable. Severe stress conditions (e.g. 2 days at 50° C.) wereneeded to generate measurable differences between the tested samples.Under these conditions, samples of combinations #9 and #10 seem toaggregate more than other samples.

The most discriminating factors for stability between the proteins arethe freeze-thaw cycles and increased temperature. Taking into accountthe very stringent stress factor of incubating at 50° C., theT366K/L351E,Y349E (combi.#4) and T366K,L351K/L351D,Y349E (combi.#11)variants are the two most stable proteins within panel, closely followedby T366K, L351K/L351D, Y349D (combi.#10) and T366K,L351K/L351D,L368E(combi.#12).

Example 21 Native MS on Ratio Experiments; Transfection Ratio's from 1:5to 5:1

To become more knowledgeable about the behavior of the CH3 mutated IgGsin skewed transfection mixtures, in particular about theT366K/L351K/L351D′:L368E′ combination (from now on dubbed KK/DE DEKK), amore elaborate ratio experiment was conducted.

Previously used antibody VH regions with known ability to pair with thecommon light chain IGKV1-39 were used for recloning into constructs 1,2, 68 and 69, resulting in vectors I-V of Table 17, Vectors I-V, eachcontaining nucleic acid sequences encoding the common human light chainas well as an Ig heavy chain with different CH3 region and differentantigen specificity, were subsequently transfected into cells withdifferent transfection ratios as indicated in Table 18. Results areshown in FIG. 23.

TABLE 17 VH Antigen VH mass Merus Cloned in Vector gene specificity (Da)designation construct # I IGHV Fibrinogen 12794 MF1122 69 3.30 (A)(L351D, L368E) II IGHV RSV (C) 13941 MF2729 69 3.23 (L351D, L368E) IIIIGHV Tetanus (B) 13703 MF1337 68 1.08 (T366K, L351K) IV IGHV Fibrinogen12794 MF1122  1 3.30 (A) (E356K, D399K) V IGHV Tetanus (B) 13703 MF1337 2 1.08 (K392D, K409D)

TABLE 18 Transfection nr vectors ratio 1 I and III 5:1 2 I and III 3:1 3I and III 1:1 4 I and III 1:3 5 I and III 1:5 6 II and III 5:1 7 II andIII 3:1* 8 II and III 1:1 9 II and III 1:3 10 II and III 1:5 11 IV and V5:1 12 IV and V 3:1 13 IV and V 1:1 14 IV and V 1:3 15 IV and V 1:5 *dueto a technical error, this sample has not been measured.

FIGS. 23A and B show that for the DEKK combination of mutations, when anexcess of A or C is present (A or C are on the ‘DE side’ and B is on the‘KK side’), AB or BC is formed but the surplus of A or C is present as amixture of both homodimers and half bodies in all cases. However, whenan excess of B is present (B is on the ‘KK side’ and A or C are on the‘DE side’), there is a clear difference. AB or BC is still formed butthe surplus of B is essentially absent as homodimer and only half bodiesare formed. Percentages were again measured by peak height Nota bene:peaks detected in the range of 2% or lower are below the threshold ofwhat the nMS technology as applied can accurately measure. Measurementsof <2% are therefore regarded to be within the noise level of analysisand therefore ignored. It is striking that the excess of B results inhigh percentages of half body B only. Especially at the 1:3 and 1:5ratios of A:B, high percentages of half body B were observed (FIGS. 23Aand 23B) in the absence of homodimer BB, indicating that the CH3mutations of the KK-side disfavour homodimerization. The absence ofhomodimers offers a crucial advantage, as this ‘KK side’ of the DEKKcombination can be chosen to incorporate a specificity that may haveknown adverse effects when present as a homodimer (for example cMET orCD3 antibodies are known to have undesired adverse side effects whenpresent as bivalent homodimers in therapeutic compositions).

The observed findings for the different ratio's of DE:KK are in contrastto the control charge reversal CH3 mutations in vectors IV and V. FIG.23C shows that for the E356K:D399K/K392D′:K409D′ combination ofmutations when an excess of A is present (A is on the ‘K392D:K409Dside’), the surplus of A is present as a mixture of both homodimers andhalf bodies in all cases, but also when an excess of B is present (B ison the ‘E356K:D399K side’), the surplus of B is present as a mixture ofboth homodimers and half bodies in all cases. Even at the higher ratios1:3 and 1:5 no half bodies B are observed although homodimers arepresent, indicating that the E356K:D399K side does not disfavourhomodimerization as much as the KK-side of the DEKK combination.

Taken together, the DEKK combination of mutations offers a clear benefitover the charge reversal CH3 mutations, in that one of the chains of theheterodimer does not form homodimers.

Example 22 Varieties of Mixtures Using the DEKK Combination

As it was demonstrated that the DEKK combination of mutations drives theformation of bispecific IgG molecules (‘AB’) with high purity, we nextexplored the feasibility of controlled production of more complexantibody mixtures from one cell, such as ‘AB and AA’ or ‘AB and AC’mixtures. Previously used model Fabs were incorporated in vectors thatcontain either the ‘DE construct’ or the ‘KK construct’ and variouscombinations of these vectors were co-expressed to create mixtures, todemonstrate the versatility of the technology. Model Fabs MF1337(tetanus toxoid), MF1122 (fibrinogen) and MF1025 (thyroglobulin) werechosen based on their overall stable behaviour, good expression levelsand mass differences between the IgGs containing these Fabs (see Table19)

TABLE 19 Specificity Fab name IgG mass Δ-mass MF1122 Tetanus (A)(MF)*1337 146747.03 +1842.05 Fibrinogen (B) (MF)1122 144904.98 0Thyroglobulin (C) (MF)1025 144259.87 −645.11 *MF = Merus Fab,designations such as MF1337 and 1337 are both used interchangeably.

TABLE 20 Transfection schedule: Tr. Heavy Heavy Heavy Tr. # chain 1chain 2 chain 3 ratio Expected species (%) Observed species (%) 11337-KK 1122-DE 1025-DE 2:1:1 AB (50%) AC (50%) AB (43%) AC (57%) 21337-DE 1122-KK 1025-KK 2:1:1 AB (50%) AC (50%) AB (40%) AC (54%) AA(6%) 3 1337-KK 1122-DE 1025-KK 1:2:1 AB (50%) BC (50%) AB (54%) BC (46%)4 1337-KK 1122-KK 1025-DE 1:1:2 AC (50%) BC (50%) AC (66%) BC (33%) CC(1%) 5 1337-KK 1337-DE 1122-DE 2:1:1 AA (50%) AB (50%) AA (57%) AB (43%)6 1337-KK 1122-KK 1122-DE 1:1:2 AB (50%) BB (50%) AB (75%) BB (25%) 71337-KK 1337-DE 1025-DE 2:1:1 AA (50%) AC (50%) AA (46%) AC (54%) 81337-KK 1025-KK 1025-DE 1:1:2 AC (50%) CC (50%) AC (60%) CC (40%) 91337-KK 1122-DE 1:1 AB (100%) AB (>98%) 10 1337-KK 1025-DE 1:1 AC (100%)AC (>98%) 11 1122-KK 1025-DE 1:1 BC (100%) AC (>98%)

SDS-PAGE analysis demonstrated that most samples consisted ofpredominantly full IgGs and in some cases half bodies were present atsmall percentages. Furthermore, many of the samples showed two bands atca. 150 kDa on non-reduced gels, reflecting the presence of two distinctIgG species in the sample. Also on the reduced gels, two heavy chainbands were visible in some samples (data not shown).

Native MS was performed on all samples and the percentages of observedspecies were calculated based on peak height (% of observed species inTable 20). Results are presented in FIG. 24. In all eight samples wherethree heavy chains were co-expressed, two main peaks were observed whichcorresponded to the expected species. In two of these samples(transfections 2 and 4), and in transfection 11, a small amount ofcontaminating DE-DE homodimer was observed. Half bodies were detected invery small amounts in most of the samples (less than 2%), which are notproblematic as they can be easily separated from the full length IgGfraction as discussed previously. After nMS it was discovered that theobserved mass of the IgG in sample 11 corresponded to a differentspecies than expected, and it was concluded that this was due to antransfection error, i.e. in sample 11 apparently 1025-DE wasco-transfected with 1337-KK instead of 1122-KK.

The IgG samples were further tested in a sandwich ELISA to confirm thefunctional presence of the desired specificities. Coating of ELISAplates was done with fibrinogen thyroglobulin and detection wasperformed with fluorescein-labelled thyroglobulin or—tetanus toxoid. Thedetection antigens were labelled with fluorescein (Pierce NHS-fluorescinAntibody Labeling kit, cat. #53029) according to the manufacturer'sinstructions. Fluorescein-labeled antigens could subsequently bedetected by a FITC-conjugated anti-fluorescein antibody (Rochediagnostics, cat. #11426346910). Results of the bispecific ELISA (OD450values) are summarized in Table 21. The greyed cells indicate theexpected species for each transfection. Generally, the results meet theexpected outcome with view exceptions as indicated in italic or bold. Intransfections 1-3, the supposed ‘negative’ well for species BC (tr. #1and 2) or AC (tr. #3) demonstrated a significant background signal. Itis known from previous studies that bispecific ELISAs may suffer fromhigh background levels. These background levels may also be caused bythe potential presence of half-bodies in the sample. Of note is that theresults of bispecific ELISA indeed confirmed that an error had occurredtransfection #11, as the species AC (bold value) was detected ratherthan BC.

TABLE 21 OD450 values from bispecific ELISA

Example 23 Improved Mixtures of Two Bispecific Antibodies Recognizing 4Different Epitopes (AB and CD) from a Single Cell

In example 12 it was hypothesized that mixtures resulting fromtransfections ZA or ZB are expected to become problematic whentransferred to larger scale production, as knob-into-hole variants arereported to be unstable and it cannot be excluded that CH3 domainscomprising a ‘knob’ or a ‘hole’ will dimerize with charge-engineered CH3domains. As it was demonstrated in the above examples that novel chargepair mutants have been found that preferentially driveheterodimerization with virtually no formation of homodimers, CH3domain-comprising polypeptide chains comprising these novel charge pairmutants can be expressed in cells together with previously knowncharge-engineered CH3 domain-comprising polypeptide chains orpotentially with SEED bodies, and are likely to result in thepreferential formation of two bispecific molecules only.

From the above examples it was clear that the DEKK combination ofmutations is excellent for the production of ore bispecific (AB) or twobispecifics (AB plus AC) by clonal cells where dimerization of the heavychains is driven by the CH3 domains. However, using only one vector setof complementary CH3 mutations limits the number of possibilities ofmixture-varieties that can be produced. It would be possible to producemore complex mixtures of IgGs and/or bispecifics, such as ‘AB and CD’ or‘AB and CC’ mixtures if a second. ‘orthogonal’ vector set could be usedin combination with DEKK. When combining two vector sets, an importantrequirement is that the heavy chains expressed from the two differentsets of CH3 engineered vectors cannot make ‘crossed’ dimers, which isthat the heavy chains produced by one of the vector sets dimerize intofull IgG with heavy chains expressed by the other vector set.

To test for such potential formation of ‘crossed.’ dimers, an in silicoanalysis was performed using HADDOCK to obtain further insights whetherpossible pairing between wildtype CH3 domains and CH3 domains containingDE- or KK-mutations would occur. Similarly, potential pairings betweenwildtype CH3 domains and CH3 domains containing E356K,D399K orK392D,K409D mutations were analyzed, as well as potential pairingsbetween wildtype CH3 domains and CH3 domains containing knob-into-holemutations and any combination of the above. Combinations of CH3-mutantsthat were analyzed in HADDOCK are listed in Table 22 and the resultingHADDOCK scores are summarized in FIG. 25.

TABLE 22 CH3 variants analyzed in HADDOCK, with one letter codes forassigned for each CH3-variant carrying heavy chain. One letter code CH3combination Mutations in HADDOCK DEKK Chain 1: T366K, L351K A Chain 2:L351D, L368E B Wildtype (WT) Chain 1: none C* Chain 2: none D* Chargereversal Chain 1: K392D, K409D A/C** (CR) Chain 2: E356K, D399K B/D**Knob-into-hole Chain 1: T366W C (KIH) Chain 2: T366S, L368A, Y407V D*Wildtype chains are designated ‘C’ and ‘D’ for matters of consistency;**The charge reversal variants are designated ‘A and B’ when combinedwith knob-into-hole variants, and are designated ‘C and D’ when combinedwith DE/KK variants.

FIG. 25 shows that, based on these HADDOCK predictions, combining theCH3 combinations of DEKK with charge reversal CH3 combinations is mostlikely to be successful in forming the desired combination of twobispecifics (AB and CD) without contaminating by-products (especiallyAC, AD, BC, BD) when co-transfected in a single cell. As can be seenfrom FIG. 25, these undesired bispecific species AC, AD, BC, and BD haverelatively high HADDOCK scores, whereas the desired AB and CD specieshave the lowest HADDOCK scores. Of course, when either the CH3combinations of DEKK or charge reversal will be put into a constructcarrying the same specificity (e.g. ‘C’ on the DE-side, ‘C’ on theKK-side, ‘A’ On the E356K,D399K-side and ‘B’ on the E356K,D399K-side, or‘A’ on the DE-side, ‘B’ on the KK-side, on the E356K,D399K-side and ‘C’on the E356K,D399K-side) this will result in the production ofpredominantly CC and AB upon co-expression in a cell.

In contrast, when looking at the predictions for co-expressing DEKK withwildtype, can be seen that the HADDOCK scores for AC and AD are lowerthan the HADDOCK score for CD, which indicates that AC and AD are verylikely contaminants when trying to produce a mixture of AB and CD byco-expression of vectors encoding for CH3 combinations of DEKK togetherwith vectors encoding wildtype CH3. Lastly, the predictions forco-expressing either DEKK or charge reversal variants together with theknob-into-hole variants results in undesired bispecific variants withrelatively low HADDOCK scores, i.e. a high likelihood that theseundesired species will be produced upon co-expression.

It is thus concluded that combining the CH3 combinations of DEKK withcharge reversal CH3 combinations (E356K,D399K/K392′D,K409D′) is ideallysuited for obtaining essentially pure ‘AB and CD’ and/or ‘AB and CC’mixtures of antibodies.

Next, mixtures of 2 bispecifics recognizing 4 targets/epitopes: (AB andCD) and mixtures of one bispecific and 1 monospecific antibodyrecognizing 3 targets/epitopes (AB and CC) were created by putting theabove into practice. These mixtures were created using 4 different. VHsthat are all capable of pairing with the common light chain IGVK1-39,but the individual VH/VL combinations all have different specificities.To enable native MS analysis, the mass difference between the (expected)species has to be sufficient, i.e. >190 Da. Four individual VHs havebeen selected and the masses of these were such that the expectedspecies upon co-transfection could be identified and separated by nMS.Furthermore, the mass differences between the 4 selected VHs are alsolarge enough to identify most of the possible contaminants in themixtures, in addition to the two desired species. Selected VHs arelisted in Table 23.

TABLE 23 VH (target) Mass as wt IgG A (RTK1) 146736.78 B (Tetanustoxoid) 146106.20 C (Fibrinogen) 144904.98 D (RTK2) 145421.37

The 4 different VHs were cloned into vectors containing the ‘DE’ or ‘KK’constructs or the charge reversal constructs, and severalco-transfections were performed as indicated in Table 24. NB: as always,all vectors also contained the nucleic acid encoding the common lightchain IGKV1-39. As previously indicated, when combining two vector sets,an important requirement is that the heavy chains expressed from the twodifferent sets of CH3 engineered vectors cannot make ‘crossed’ dimers,which is that the heavy chains produced by one of the vector setsdimerize into full IgG with heavy chains expressed by the other vectorset. To test for such potential formation of ‘crossed’ dimers betweenheavy chains containing charge reversal mutations and heavy chainscontaining DE or KK mutations, control transfections were performed.

TABLE 24 1^(st) VH/ 2^(nd) VH/ Tr. # construct # construct # Expectedspecies 1 D/68 A/68 mismatch ‘KK’ with ‘KK’; Mostly half-bodies expected2 D/68 A/69 match ‘KK’ with ‘DE’; AD product expected 3 D/68 A/1Expected mismatch ‘KK’ with ‘E356K:D399K’ 4 D/68 A/2 Expected mismatch‘KK’ with ‘K392D:K409D’ 5 D/69 A/68 match ‘DE’ with ‘KK’; AD productexpected 6 D/69 A/69 mismatch ‘DE’ with ‘DE’; mixture of half- bodies,AA, AD and DD expected 7 D/69 A/1 Expected mismatch ‘DE’ with‘E356K:D399K’ 8 D/69 A/2 Expected mismatch ‘DE’ with ‘K392D:K409D’1^(st) VH/ 2^(nd) VH/ 3^(rd) VH/ 4^(th) VH/ Expected Tr. # construct #construct # construct # construct # species  9 A/68 B/69 C/1 D/2 AB andCD 10 A/68 A/69 C/1 D/2 AA and CD 11 A/68 B/69 C/1 C/2 AB and CC

Table 25 provides a further overview of masses of the expected species,and the possible contaminants, of transfections #9-11 of Table 24.

TABLE 25 For each of transfections #9-11, the species are sorted bymass, mass difference is calculated with the mass above.

Grey cells: expected (and desired) species; italics: mass difference toosmall to separate in nMS analysis. *Speices: single letters representhalf-bodies; two-letter code intact IgG.

All purified protein samples obtained from transfections #1-11 wereanalyzed on SDS-PAGE, and three control samples were included (FIG. 26).In addition, nMS analysis was performed on protein samples fromtransfections #9-11 to identify all species in the samples. As can beseen from FIG. 26, transfections #3 and #4 resulted in the expectedmismatch between ‘KK’ constructs and either ‘E356K:D399K’ or‘K392D:K409D’ and the amount of half bodies in protein samples fromthese transfections exceeded the amount of full IgG molecules.Transfections #7 and #8 resulted in protein samples wherein both halfbodies and full IgG is present in about equal amounts. However, fromSDS-PAGE it cannot be deduced whether the full. IgG represents a DE/DEdimer, a DE/E356K:D399K dimer or a DE/K392D:K409D dimer. Remarkably,virtually no half bodies were observed in samples from transfections#9-11.

In FIG. 27, the nMS analysis of transfections #9 and #11 are presented.Percentages of expected species and contaminating species werecalculated by peak height. It was demonstrated that, for transfection#9, the expected species ‘AB and CD’ are represented for 97% in themixture (30% AB and 67% CD) whereas only as little of about 3% ofcontaminating BD is present (FIG. 27A). For transfection #11, theexpected species ‘AB and CC’ are represented for 94% in the mixture (33%AB and 61% CC) whereas only as little of about 6% of contaminating BC(4.1%) and AC (1.8%) is present (FIG. 27B). These data show that it isindeed possible to produce more complex mixtures of IgGs and/orbispecifics, such as ‘AB and CD’ or ‘AB and CC’ mixtures when a second‘orthogonal’ vector set is used in combination with DEKK. Combination ofthe charge reversal constructs together with the DEKK constructs resultsin only very limited formation of ‘crossed’ dimers. By adjusting thetransfection ratio's it is expected that the low percentages of thesecontaminating by-products can be even further reduced.

Example 24 Single Dose Pharmacokinetic Study in Mice

To study the pharmacokinetic (pK) behavior of bispecific antibodiescarrying the DEKK combination of mutations in their CH3 regions, in thisstudy the pK parameters for three different IgG batches were determinedand compared. The three IgG batches included 1) wildtype anti-tetanustoxoid parental antibody 1337:1337 (two MF1337 Fabs on a wildtype Fcbackbone); 2) wildtype anti-tetanus toxoid parental antibody 1516:1516(two MF1516 Fabs on a wildtype Fc backbone); 3) CH3 engineeredbispecific anti-tetanus toxoid antibody 1516:1337 that carries the DEKKcombination of mutations in its Fc region (MF1516 Fab on DE-side, MF1337Fab on KK-side).

The parental antibodies 1337:1337 and 1516:1516 were chosen asspecificities to be included in the DEKK-bispecific product, as it wasknown based on previous studies that no pre-dose serum response againstthese antibodies was present in several mice strains, NB: the presenceof a pre-dose serum response would of course invalidate the study. Inaddition, there is sufficient mass difference between the parentalantibodies to enable the identification of 1337:1337 (wt. Fe)_(;)1516:1337 (DEKK Fe) and 1516:1516 (wt Fc) species by nMS. The three IgGbatches were prepared as previously described, but the DNA used fortransfection was made using an endo-free maxiprep kit to ensure that theamount of endotoxins is as low as possible. The batches weresubsequently tested for protein concentration, aggregate levels,endotoxin levels and percentage bispecific product. It was demonstratedthat the acceptance criteria for subsequent use of the IgG batches in apK study were met, i.e. the IgG concentration after gel filtrationwas >0.3 mg/ml, aggregate levels were <5%, endotoxin levels were <3EU/mg protein and the DEKK batch contained >90% bispecific IgG. Nativemass spectrometry of the gel filtrated samples showed that the expectedspecies were present in high percentages. In sample 1516:1337 a smallamount of the DE:DE homodimer is detected, which is estimated to be ca.2% (FIG. 28). It was concluded that the 3 IgG batches are qualified tobe used in the pK study.

For comparison of pK parameters between the three batches, 3 groups offemale C57BL/6J mice (Harlan, The Netherlands) were dosed at 1 mg/kghuman IgG (5 ml/kg immunoglobulin solution/kg body weight). At dosingtime, the animals were between 7-8 weeks of age and had a body weight ofabout 18-20 grams. Blood samples were collected pre-dose and at 15, 60minutes, and 2, 4, 8, 24, 48, 96, 168, 268 and 336 h after dosing. Serumsamples were prepared and stored at <−20° C. until analysis. Each groupconsisted of 3 subgroups of 4 mice, i.e. 12 mice/group. From each mice 6time points were sampled. The welfare of the animals was maintained inaccordance with the general principles governing the use of animals inexperiments of the European Communities (Directive 86/609/EEC) and Dutchlegislation (The Experiments on Animals Act, 1997). This study was alsoperformed in compliance with the Standards for Humane Care and Use ofLaboratory Animals, as issued by the Office of Laboratory Animal Welfareof the U.S. National Institutes of Health under identification number45859-01 (expiration date: 30 Apr. 2015).

Mice of Group 1 received the full length monospecific IgG 1516:1516antibody (triangles); Mice of Group 2 received the full lengthmonospecific IgG 1337:1337 antibody (squares); Mice of Group 3 receivedthe full length bispecific IgG 1516:1337 antibody (diamonds), with DEKKengineered CH3 regions (1516 on the DE-side and 1337 on the KK-side).

An ELISA assay was applied for the quantitative analysis of monoclonalhuman antibodies in mouse serum using a quantitative human IgG ELISA(ZeptoMetrix, NY USA; ELISA kit nr. 0801182). Briefly, the ELISA assayis based on the principle that the human monoclonal antibody binds toanti-human IgG coated in a 96-wells ELISA plate. Bound antibody wassubsequently visualized using a polyclonal antihuman IgG antibodyconjugated with horseradish peroxidase (IMP). The optical density (OD)of each well is directly proportional to the amount of antibody in theserum sample. Results are shown in FIG. 29, and it was observed thatserum levels of both the bispecific full length IgG antibody carryingthe DEKK combination of mutations and its parental monospecificantibodies are strikingly similar. It is concluded that the CH3mutations as present in the DEKK-bispecific antibody does not alterstability nor half life, and the DEKK variant is behaving as wildtypeIgG.

REFERENCES

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The invention claimed is:
 1. A method for producing a heterodimericantibody from a single cell, wherein said antibody comprises two heavychains with CH3 domains that are capable of forming an interface, saidmethod comprising: a. providing a host cell comprising (i) a firstnucleic acid molecule encoding a 1^(st) antibody heavy chain comprisingat least one substitution of a neutral amino acid residue in the CH3domain by a positively charged amino acid residue, and (ii) a secondnucleic acid molecule encoding a 2^(nd) antibody heavy chain comprisingat least one substitution of a neutral amino acid residue in the CH3domain by a negatively charged amino acid residue; b. culturing saidhost cell and allowing for expression of said two nucleic acid moleculesto produce said 1^(st) antibody heavy chain and said 2^(nd) antibodyheavy chain, wherein the at least one positively charged amino acidresidue substituted in the CH3 domain of said 1^(st) antibody heavychain interacts with the at least one negatively charged amino acidresidue substituted in the CH3 domain of said 2^(nd) antibody heavychain in the interface between said 1^(st) and 2^(nd) antibody heavychains to produce a heterodimeric antibody; and c. harvesting saidheterodimeric antibody from the culture.
 2. The method of claim 1,further comprising providing said host cell with a nucleic acid moleculeencoding a common light chain.
 3. The method of claim 1, wherein said1^(st)-antibody heavy chain comprises a substitution of the amino acidresidue at position 366 in the CH3 domain by a lysine (K) residue, andwherein said 2^(nd) antibody heavy chain comprises a substitution of theamino acid residue at position 351 in the CH3 domain by an aspartic acid(D) residue.
 4. The method of any one of claims 1-3, wherein the CH3domain of said 1^(st) antibody heavy chain further comprises asubstitution of the amino acid residue at position 351 by a lysine (K)residue.
 5. The method of claim 4, wherein the CH3 domain of said 2^(nd)antibody heavy chain further comprises amino acid substitution(s)selected from the group consisting of (i) a substitution of the aminoacid residue at position 349 by a glutamic acid (E) residue; (ii) asubstitution of the amino acid residue at position 349 by an asparticacid (D) residue; (iii) a substitution of the amino acid residue atposition 368 by a glutamic acid (E) residue; (iv) a substitution of theamino acid residue at position 349 by an aspartic acid (D) residue, anda substitution of the amino acid residue at position 368 by a glutamicacid (E) residue; and (v) substitution of the amino acid residues atpositions 349 and 355 by aspartic acid (D) residues.
 6. The method ofclaim 5, wherein the CH3 domain of the 2^(nd) antibody heavy chaincomprises the substitution of the amino acid residue at position 368 bya glutamic acid (E) residue.
 7. The method according to claim 1, whereinthe presence of contaminating homodimers is less than 5%, preferablyless than 2%, more preferably less than 1%, and most preferablycontaminating homodimers are essentially absent.
 8. The method of claim1, wherein each variable region of the 1^(st) and 2^(nd) antibody heavychains recognizes a different target epitope.
 9. The method of claim 8,wherein the different target epitopes are located on the same targetmolecule.
 10. The method of claim 9, wherein the target molecule is asoluble molecule.
 11. The method of claim 9, wherein the target moleculeis a membrane-bound molecule.
 12. The method of claim 8, wherein thedifferent target epitopes are located on different target molecules. 13.The method of claim 12, wherein the different target molecules areexpressed on the same cells.
 14. The method of claim 12, wherein thedifferent target molecules are expressed on different cells.
 15. Themethod of claim 12, wherein the different target molecules are solublemolecules.
 16. The method of claim 12, wherein one target molecule is asoluble molecule whereas the second target molecule is a membrane boundmolecule.
 17. The method of claim 8, wherein at least one of said targetepitopes is located on a tumor cell.
 18. The method of claim 8, whereinat least one of said target epitopes is located on an effector cell. 19.The method of claim 18, wherein said effector cell is an NK cell, a Tcell, a B cell, a monocyte, a macrophage, a dendritic cell or aneutrophilic granulocyte.
 20. The method of claim 18, wherein saidtarget epitope is located on a CD3, CD16, CD25, CD28, CD64, CD89, NKG2Dor a NKp46 molecule.
 21. The method according claim 1, wherein saidheterodimeric antibody is human IgG.
 22. A heterodimeric antibodyobtained by the method of claim
 1. 23. A heterodimeric antibodyaccording to claim 22, wherein said heterodimeric antibody binds todifferent epitopes on the same antigen or to different epitopes ondifferent antigens.
 24. A heterodimeric antibody according to claim 22,wherein said antibody is human IgG.
 25. A heterodimeric antibodycomprising two CH3 domains, wherein one of said two CH3 domainscomprises an amino acid substitution at position 351 by an aspartic acid(D) and an amino acid substitution at position 368 by a glutamic acid(E), and wherein the other of said two CH3 domains comprises an aminoacid substitution at position 366 by a lysine (K) and an amino acidsubstitution at position 351 by a lysine (K).
 26. A recombinant hostcell comprising nucleic acid sequences encoding at least a 1st and2^(nd) antibody heavy chain, wherein the CH3 domain of said 1^(st)antibody heavy chain comprises at least one substitution of a neutralamino acid residue by a positively charged amino acid residue, andwherein the CH3 domain of said 2^(nd) antibody heavy chain comprises atleast one substitution of a neutral amino acid residue by a negativelycharged amino acid residue.
 27. A recombinant host cell according toclaim 26, wherein said host cell further comprises a nucleic acidsequence encoding a common light chain.
 28. A pharmaceutical compositioncomprising a heterodimeric antibody according to claim 22 and apharmaceutically acceptable carrier.
 29. A pharmaceutical compositioncomprising a heterodimeric antibody according to claim 25, and apharmaceutically acceptable carrier.
 30. A method for making a host cellfor production of a heterodimeric antibody, the method comprising thestep of introducing into said host cell nucleic acid molecules encodingat least a 1^(st) and 2^(nd) antibody heavy chain, wherein the CH3domain of said 1^(st) antibody heavy chain comprises at least onesubstitution of a neutral amino acid residue by a positively chargedamino acid residue and wherein the CH3 domain of said 2^(nd) antibodyheavy chain comprises at least one substitution of a neutral amino acidresidue by a negatively charged amino acid residue, wherein said nucleicacid sequences are introduced consecutively or concomitantly.
 31. Amethod according to claim 30, further comprising the step of introducinginto said host cell a nucleic acid sequence encoding a common lightchain.
 32. The method according to claim 21 wherein antibody is humanIgG1.